Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to ...initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.
The initial observation that p53 accumulation might serve as a surrogate biomarker for TP53 mutation has been the cornerstone for vast translational efforts aimed at validating its clinical use for ...the diagnosis, prognosis, and treatment of cancer. Early on, it was realized that accurate evaluation of p53 status and function could not be achieved through protein-expression analysis only. As our understanding of the p53 pathway has evolved and more sophisticated methods for assessment of p53 functional integrity have become available, the clinical and molecular epidemiological implications of p53 abnormalities in cancers are being revealed. They include diagnostic testing for germline p53 mutations, and the assessment of selected p53 mutations as biomarkers of carcinogen exposure and cancer risk and prognosis. Here, we describe the strengths and limitations of the most frequently used techniques for determination of p53 status in tumors, as well as the most remarkable latest findings relating to its clinical and epidemiological value.
Most human pre-mRNA transcripts are alternatively spliced, but the significance and fine-tuning of alternative splicing in different biological processes is only starting to be understood. SRSF3 ...(SRp20) is a member of a highly conserved family of splicing factors that have critical roles in key biological processes, including tumor progression. Here, we show that SRSF3 regulates cellular senescence, a p53-mediated process to suppress tumorigenesis, through TP53 alternative splicing. Downregulation of SRSF3 was observed in normal human fibroblasts undergoing replicative senescence, and was associated with the upregulation of p53β, an alternatively spliced isoform of p53 that promotes p53-mediated senescence. Knockdown of SRSF3 by short interfering RNA (siRNA) in early-passage fibroblasts induced senescence, which was associated with elevated expression of p53β at mRNA and protein levels. Knockdown of p53 partially rescued SRSF3-knockdown-induced senescence, suggesting that SRSF3 acts on p53-mediated cellular senescence. RNA pulldown assays demonstrated that SRSF3 binds to an alternatively spliced exon uniquely included in p53β mRNA through the consensus SRSF3-binding sequences. RNA crosslinking and immunoprecipitation assays (CLIP) also showed that SRSF3 in vivo binds to endogenous p53 pre-mRNA at the region containing the p53β-unique exon. Splicing assays using a transfected TP53 minigene in combination with siRNA knockdown of SRSF3 showed that SRSF3 functions to inhibit the inclusion of the p53β-unique exon in splicing of p53 pre-mRNA. These data suggest that downregulation of SRSF3 represents an endogenous mechanism for cellular senescence that directly regulates the TP53 alternative splicing to generate p53β. This study uncovers the role for general splicing machinery in tumorigenesis, and suggests that SRSF3 is a direct regulator of p53.
The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes ...in collagen—including dentinal collagen—is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)–derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.
An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental ...models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10–15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses.
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•SAEC-A3 and SAEC-H7 are new monoclonal ES cell lines from gilthead seabream embryos.•SAEC cell lines proliferate indefinitely by adapting to different culture conditions.•These cell lines generate embryoid bodies and are sensitive to all-trans retinoic acid.•Daily expression of pcna showed significant rhythms of proliferation in SAEC-H7.•Transfection of cell lines indicates they represent useful biotechnological tools.
Polyamines are small organic and basic polycations that perform essential regulatory functions in all living organisms. Fluctuations in polyamine content have been observed to occur during growth, ...development and under stress conditions, implying that polyamines play pivotal roles in diverse cellular and physiological processes. To achieve polyamine homeostasis, the entire metabolic pathway is subjected to a fine-tuned regulation of its biosynthetic and catabolic genes and enzymes. In this review, we describe and discuss the most important mechanisms implicated in the translational and post-translational regulation of polyamine metabolic enzymes in plants. At the translational level, we emphasize the role of polyamines in the modulation of upstream open reading frame (uORF) activities that control the translation of polyamine biosynthetic and catabolic mRNAs. At the post-translational level, different aspects of the regulation of polyamine metabolic proteins are depicted, such as the proteolytic activation of enzyme precursors, the importance of dimerization in protein stability as well as in protein intracellular localization.
•Elements in the 5′-UTR negatively regulate translation of polyamine metabolic mRNAs.•Polyamines modulate uORF activity to control translation of their metabolic mRNAs.•Dimerization impacts the localization and activity of polyamine metabolic proteins.•Polyamine metabolic proteins with PEST sequences are targeted to the 26S-proteasome.•Proteolytic cleavage of enzyme precursors is necessary for polyamine biosynthesis.
Sin embargo, antes de llegar a este punto es necesario analizar el cumplimiente los protocolos de actuación existentes. Desde hace algunos años, al introducirnos en el campe estudie las emergencias, ...se remarcan las diferencias entre el enfoque tradicionae gestión y el modelo actual, puesto que han supuesto cambios importantes. Por otra parte, si la Psicología de las Emergencias y los Desastres es la rama de la psicología que abarca el estudiel comportamiento y el mode reacción tanto individual como grupal en las diferentes fases de una situación de emergencia esastre y ese comportamiento y mode reacción, en gran medida, se puede predecir y extrapolar del aprendizaje de situaciones anteriores, se pone de manifiesto que se debe actuar de modo preventivo y proactivo para evitar, si se puede, que las más que posibles “reacciones normales ante situaciones anormales” vayan a más e interfieran en el normal funcionamientel individuo en su entorno, causando malestar individual y social. Es una intervención destinada a modificar las condiciones generadoras de riesgo, con el fin de reducir los niveles del mismo, minimizándolos hasta donde sea posible.
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