ß-Catenin Regulation during the Cell Cycle: Implications in G2/M and Apoptosis

ß-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. ß-Catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems ß-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of ß-Catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous ß-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase¿synchronized epithelial cells. ß-Catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of ß-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not ß-catenin, is detected in M phase. Interestingly, overexpression of a stable form of ß-catenin, or inhibition of endogenous ß-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for ß-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.

Fusion of the human gene for the polyubiquitination co-effector UEV-1 with Kua, a newly identified gene

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).

Minimizing recombinations in consensus networks for phylogeographic studies

Background: We address the problem of studying recombinational variations in (human) populations. In this paper, our focus is on one computational aspect of the general task: Given two networks G1 and G2, with both mutation and recombination events, defined on overlapping sets of extant units the objective is to compute a consensus network G3 with minimum number of additional recombinations. We describe a polynomial time algorithm with a guarantee that the number of computed new recombination events is within ϵ = sz(G1, G2) (function sz is a well-behaved function of the sizes and topologies of G1 and G2) of the optimal number of recombinations. To date, this is the best known result for a network consensus problem.Results: Although the network consensus problem can be applied to a variety of domains, here we focus on structure of human populations. With our preliminary analysis on a segment of the human Chromosome X data we are able to infer ancient recombinations, population-specific recombinations and more, which also support the widely accepted 'Out of Africa' model. These results have been verified independently using traditional manual procedures. To the best of our knowledge, this is the first recombinations-based characterization of human populations. Conclusion: We show that our mathematical model identifies recombination spots in the individual haplotypes; the aggregate of these spots over a set of haplotypes defines a recombinational landscape that has enough signal to detect continental as well as population divide based on a short segment of Chromosome X. In particular, we are able to infer ancient recombinations, population-specific recombinations and more, which also support the widely accepted 'Out of Africa' model. The agreement with mutation-based analysis can be viewed as an indirect validation of our results and the model. Since the model in principle gives us more information embedded in the networks, in our future work, we plan to investigate more non-traditional questions via these structures computed by our methodology.

The transcriptional co-activator PCAF regulates cdk2 activity.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

The cell cycle–regulated genes of schizosaccharomyces pombe

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S.pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

Constructos personales y perfil sintomático en la etapa del climaterio: Un estudio exploratorio.

En este estudio se exploraron algunas características cognitivas y sintomáticas en un grupo de mujeres en etapa de climaterio, que van a consultar a centros de salud ( G1) , en un grupo de mujeres en etapa de climaterio, que no consultaron de forma habitual ( G2 ) y en un grupo de mujeres adultas, de menor edad ( G3 ) . Todas las mujeres fueron evaluadas a través de la Técnica de Rejilla Interpersonal (TRI) y de la escala de síntomas SCL-90-R. Sólo se observaron diferencias significativas en el mayor aislamiento social percibido por G1 y G2 comparado con G3. No obstante, se observó que GI presenta una tendencia a diferir de G2 y G3 en varias de las dimensiones de TRI, las que insinúan una posible influencia del estereotipo social negativo ligado al climaterio en el significado de sus problemas de salud. En el SCL-90-R las diferencias significativas entre los grupos revelan la importancia de las sensaciones de malestar general en salud del G1 por encima del perfil, de perturbaciones típicas del climaterio.

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.

Lamellarin D bioconjugates I: synthesis and cellular internalization of PEG-derivatives

Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties, and improving the biological activity. Mono-, di- and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI50 than Lam-D. Furthermore, cell cycle arrest at G2 phase, and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.

Lamellarin D bioconjugates I: synthesis and cellular internalization of PEG-derivatives

Herein is reported the design and synthesis of poly(ethylene glycol) derivatives of Lamellarin D with the aim of modulating their physicochemical properties, and improving the biological activity. Mono-, di- and tri-PEG conjugates with improved solubility were obtained in 18-57% overall yields from the corresponding partially protected phenolic derivatives of Lamellarin D. Conjugates 1-9 were tested in a panel of three human tumor cell lines (MDA-MB-231 breast, A-549 lung and HT-29 colon) to evaluate their cytotoxicity. Several compounds exhibited enhanced cellular internalization, and more than 85% of the derivatives showed a lower GI50 than Lam-D. Furthermore, cell cycle arrest at G2 phase, and apoptotic cell-death pathways were determined for Lamellarin D and these derivatives.