Dynamics of DNA Replication Factories in Living Cells

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase

Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed “functional organization” of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1–28 and 111–179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins

Knocking-Down Cyclin A2 by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A2 is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A2 is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A2 and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A2 by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A2 in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A2 showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A2 and suggest that cyclin A2 is a key regulator of cell differentiation. To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells

Etude du mécanisme de dégradation de la Mitotic Kinesin like protein 2 en sortie de mitose

Le cycle cellulaire est indispensable à la vie eucaryote. En effet, tout organisme a besoin pour se reproduire, se développer ou se régénérer, de diviser ses cellules. Il est apparu que ce dispositif conceptuellement simple reposait sur une mécanistique compliquée. Je me suis intéressée à la division proprement dite, et à la régulation de MKlp2 (Mitotic Kinesin like protein 2) chez les mammifères. J ai mis au point une technique permettant de trier et d analyser des cellules en cytodiérèse par cytométrie en flux. Puis j ai observé le profil d expression de MKlp2 dans les hépatocytes. MKlp2 est détectée uniquement dans les hépatocytes en prolifération. Sa localisation est nucléaire en G2, au niveau des chromatides en prophase et en métaphase, puis sur le fuseau central en anaphase et au midbody en télophase et cytodiérèse. MKlp2 est un nouveau marqueur de prolifération hépatocytaire. Enfin, j ai étudié le mécanisme de dégradation de MKlp2 en sortie de mitose. J ai montré que la dégradation de MKlp2 dépend de l APC/CCdh1 mais sa reconnaissance n est pas dûe à un motif connu. De plus, on observe que les mutants non dégradables sont mal localisés dans la cellule.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Impaired angiogenesis and tumor development by inhibition of the mitotic kinesin Eg5.

peer reviewedKinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors

The oncolytique minute virus of mice bloks cellular proliferation :analysis of the mechanism.

info:eu-repo/semantics/nonPublishe

Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM.

Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S