Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

Minichromosome maintenance proteins 2 and 5 in non-benign epithelial ovarian tumours: relationship with cell cycle regulators and prognostic implications

Minichromosome maintenance proteins (MCM) have recently emerged as novel proliferation markers with prognostic implications in several tumour types. This is the first study investigating MCM-2 and MCM-5 immunohistochemical expression in a series of ovarian adenocarcinomas and low malignant potential (LMP) tumours aiming to determine possible associations with clinicopathological parameters, the conventional proliferation index Ki-67, cell cycle regulators (p53, p27Kip1, p21WAF1 and pRb) and patients' outcome. Immunohistochemistry was applied in a series of 43 cases of ovarian LMP tumours and 85 cases of adenocarcinomas. Survival analysis was restricted to adenocarcinomas. The median MCM-2 and MCM-5 labelling indices (LIs) were significantly higher in adenocarcinomas compared to LMP tumours (P<0.0001 for both associations). In adenocarcinomas, the levels of MCM-2 and MCM-5 increased significantly with advancing tumour stage (P=0.0052 and P=0.0180, respectively), whereas both MCM-2 and MCM-5 increased significantly with increasing tumour grade (P=0.0002 and P=0.0006, respectively) and the presence of bulky residual disease (P<0.0001 in both relationships). A strong positive correlation was established between MCM-2 or MCM-5 expression level and Ki-67 LI (P<0.0001) as well as p53 protein (P=0.0038 and P=0.0500, respectively). Moreover, MCM-2 LI was inversely correlated with p27Kip−1 LI (P=0.0068). Finally, both MCM-2 and MCM-5 were associated significantly with adverse patients' outcome in both univariate (⩾20 vs >20%, P=0.0011 and ⩾25 vs <25%, P=0.0100, respectively) and multivariate (P=0.0001 and 0.0090, respectively) analysis. An adequately powered independent group of 45 patients was used in order to validate our results in univariate survival analysis. In this group, MCM-2 and MCM-5 expression retained their prognostic significance (P<0.0001 in both relationships). In conclusion, MCM-2 and MCM-5 proteins appear to be promising as prognostic markers in patients with ovarian adenocarcinomas

Expression of minichromosome maintenance protein 2 as a marker for proliferation and prognosis in diffuse large B-cell lymphoma: a tissue microarray and clinico-pathological analysis

BACKGROUND: Minichromosome maintenance (MCM) proteins are essential for the initiation of DNA replication and have been found to be relevant markers for prognosis in a variety of tumours. The aim of this study was to assess the proliferative activity of diffuse large B-cell lymphoma (DLBCL) in tissue microarray (TMA) using one of the minichromosome maintenance proteins (Mcm2) and to explore its potential value to predict prognosis. METHODS: Immunohistochemistry for Mcm2 was performed on TMAs constructed from 302 cases of DLBCL. A monoclonal mouse antibody was used after heat induced antigen retrieval. Mcm2 expression was scored quantitatively. Positivity for Mcm2 was defined as presence of nuclear expression of Mcm2 in greater than or equal to 40 % of tumour cells. A statistical analysis was carried out of the association of Mcm2 and the clinico-pathological characteristics. RESULTS: Mcm2 expression was clearly evident in the nuclei of proliferating non-neoplastic cells and tumour cells. Positivity for Mcm2 was found in 46% (98/211) of analysable cases. A significant correlation existed between Mcm2 expression and presence of bulky disease (p = 0.003). Poor disease specific survival was observed in patients with DLBCL positive for Mcm2 expression in the univariate analysis (p = 0.0424). CONCLUSION: Mcm2 expression can be used to assess tumour proliferation and may be useful as an additional prognostic marker to refine the prediction of outcome in DLBCL

Preferential Re-Replication of Drosophila Heterochromatin in the Absence of Geminin

To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase

Empirische Deliberationsforschung

Die deliberative Demokratietheorie ist die wichtigste zeitgenössische Demokratietheorie. Das Versprechen der deliberativen Demokratietheorie, die Legitimation und die Qualität politischer Entscheidungen gleichzeitig zu erhöhen, hat dazu geführt, dass auf allen Ebenen des demokratischen Prozesses diskursive Entscheidungsverfahren unterschiedlicher Art implementiert worden sind. Die wichtige Frage lautet jedoch: Können die empirischen Verfahren die theoretischen Versprechungen wirklich einlösen? Die empirische Diskursforschung bietet hier erste Antworten. In dem Papier diskutieren wir einerseits die Herausforderungen, die aus den empirischen Studien für die Theorie resultieren. Andererseits werden die empirischen Studien selbst auf ihre Aussagekraft hin kritisch befragt. Wir argumentieren, dass die geringe Beachtung, die den Gegenständen der Deliberation bislang geschenkt wurde, ein wichtiger Grund für die disparaten Ergebnisse sein kann, die die empirischen Studien zur deliberativen Demokratietheorie bislang hervorgebracht haben. Es wird darauf hingewiesen, dass der Erfolg einer Deliberation wesentlich davon abhängt, ob die Entscheidungsgegenstände als regulative Fragen, Interessenskonflikte oder ethisch-moralische Fragen geframed werden.The theory of deliberative democracy has strongly influenced philosophical work on democracy over the past twenty years. On at least a theoretical basis, deliberative democracy offers a promising way to improve both the legitimacy and the quality of political decisions at once. This prospect has motivated political scientists and politicians all over the world to implement and analyze a multitude of deliberative forums at all levels of the democratic decision-making process. But the question remains: Can real deliberations fulfill the promise of the theory? Empirical research on deliberation may provide the answer. In our paper, we first discuss the challenges that such studies present to deliberative theory. We then take a critical look at the empirical work that has been done so far on verifying deliberative democratic theory. Comparing different studies on this topic can be quite a frustrating experience, because their results tend to be disparate and sometimes even contradictory. We argue that in order to improve this situation, empirical scientists should pay more attention to the subjects of deliberation. For example, we believe that one of the keys to a successful deliberative process lies in how the topics of the discourse are framed.1 Stand der Forschung und Erkenntnisinteresse Einführung Die normative Theorie und ihre empirische Prüfbarkeit 2 Empirische Deliberationsforschung Deliberation unter Laborbedingungen Real-World-Deliberation Real-World-Deliberationen unter Bürgern Real-World-Deliberationen in institutionalisierten Entscheidungskontexten Deliberation und Massenmedien 3 Theoretische Klarheit – empirische Verwirrung? Literatu