CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin-dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers

CDC25B overexpression stabilises centrin 2 and promotes the formation of excess centriolar foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 "foci". These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability

Incorporación de mayor valor en la cadena de la miel y productos derivados de la colmena en el Pacífico Central, Costa Rica

En este documento se ofrece una propuesta para el fortalecimiento de la cadena de valor de la miel de abeja y para la incorporación de valor agregado a los productos derivados de la colmena en la región del Pacífico Central costarricense. El proceso se llevó a cabo mediante un acuerdo de cooperación técnica entre el Ministerio de Economía, Industria y Comercio de Costa Rica (MEIC) y la Sede Subregional de la Comisión Económica para América Latina y el Caribe (CEPAL) en México, en el marco de las actividades del proyecto CEPAL-CRUSA “Fortalecimiento de dos cadenas de valor con alto potencial de encadenamientos para PYME en el Pacífico Central costarricense”.Resumen .-- Introducción .-- I. Aspectos generales de la cadena de la miel .-- II. Caracterización de la cadena de la miel en el Pacífico Central .-- III. Oportunidades para incrementar el valor agregado de la cadena .-- IV. Gobernanza .-- V. Análisis de la sostenibilidad ambiental .-- VI. Restricciones .-- VII. Buenas prácticas .-- VIII. Estrategia para fortalecer la cadena de la miel y productos derivados de la colmena a partir de la generación de productos de mayor valor agregado .-- IX. Conclusiones

Known genotoxic compounds identified with the DDR-Act-Fp reporter system amongst 1,280 pharmacologically compounds of the LOPAC library.

<p>Results are expressed as percentage of the fluorescence intensity measured in cells treated with 10μM Etoposide. ID number, compound name and putative mode of action are indicated.</p

Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model

<div><p>Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.</p></div

Additional file 2: of Gap junctions contribute to anchorage-independent clustering of breast cancer cells

Figure S2. Effect of the combination of latrunculin A and meclofenamate on the clustering of MCF7 cancer cells and on calcein transfer. (A) Variation of the area occupied by MCF7 cells during the clustering assay with cells incubated or not (n = 21) with 100 nM latrunculin A (n = 22), 300 μM meclofenamate (n = 26), or latrunculin A + meclofenamate (n = 27). Results are the mean ± SD of 4 independent experiments. Mann-Whitney non-parametric tests, except for NT versus latrunculin A + meclofenamate: unpaired two-tailed t-test at 2 h, ***p < 0.0005. (B) Transfer of calcein during the clustering assay from donor positive cells to negative cells incubated or not with different compounds as in (A). The percentage of receiver positive cells is indicated. Results are the mean ± SD of 4 independent experiments (3 replicates for each condition in each experiment). Unpaired two-tailed t-tests, at 2 h, 5 h and 10 h; differences are not statistically significant (N.S.). (PDF 575 kb

A Fluorescent assay to monitor DDR pathway activation.

<p>(A) Schematic representation of the principle of the DDR-Act-FP reporter. Upon DNA-damage dependent accumulation of p53, transcriptional activation of the p21 promoter leads to the expression of the fluorescent protein (FP). (B) Principle of the assay. A cell line stably expressing the DDR-Act-FP reporter is used to produce 3D spheroids. DDR pathway activation can be monitored globally on 2D monolayer cultured cells and on 3D spheroids grown in 96 well plates using a High Content Screening approach.</p