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Bua S.,DNA Replication Group | Bua S.,Institute Pasteur Paris | Sotiropoulou P.,Free University of Colombia | Sgarlata C.,DNA Replication Group | And 10 more authors.
Cell Cycle | Year: 2015

Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice. © 2015 Taylor & Francis Group, LLC.

Marion R.M.,Telomeres and Telomerase Group | Schotta G.,Ludwig Maximilians University of Munich | Ortega S.,Transgenic Mice Unit | Blasco M.A.,Telomeres and Telomerase Group
PLoS ONE | Year: 2011

Reprogramming of adult differentiated cells to induced pluripotent stem cells (iPS) cells has been achieved by over-expression of specific transcription factors. Nuclear reprogramming induces a series of profound changes at the telomeres of the parental differentiated cells, including a telomerase-dependent telomere elongation and the remodeling of telomeric chromatin. In particular, iPS cells show a decreased density of H4K20me3 heterochromatic mark at telomeres compared to the parental cells. Suv4-20h1 and Suv4-20h2 histone methytransferases (HMTases) are responsible for the trimethylation of H4K20 at telomeres, as cells deficient for both HMTases show decreased levels of H4K20me3 at telomeric chromatin. Here, we set to address the role of the Suv4-20h enzymes in telomere reprogramming by generating bona-fide iPS cells from mouse embryonic fibroblasts (MEFs) double null for both HMTases (Suv4-20dn MEFs). We found that Suv4-20h deficiency enhances telomere elongation during reprogramming without altering their ability to protect the chromosome ends or the efficiency of reprogramming. Moreover, teratomas generated from Suv4-20dn iPS cells also have elongated telomeres and an increased growth rate when compared to wild-type controls. These results indicate that abrogation of Suv4-20h enzymes and loss of heterochromatic mark H4K20me3 at telomeric heterochromatin facilitates telomere reprogramming and provides an increased tumorigenic potential to the resulting iPS cells. © 2011 Marión et al.

De Bonis M.L.,Telomeres and Telomerase Group | Ortega S.,Transgenic Mice Unit | Blasco M.A.,Telomeres and Telomerase Group
Stem Cell Reports | Year: 2014

The NAD-dependent deacetylase SIRT1 is involved in chromatin silencing and genome stability. Elevated SIRT1 levels in embryonic stem cells also suggest a role for SIRT1 in pluripotency. Murine SIRT1 attenuates telomere attrition in vivo and is recruited at telomeres in induced pluripotent stem cells (iPSCs). Because telomere elongation is an iPSC hallmark, we set out to study the role of SIRT1 in pluripotency in the setting of murine embryonic fibroblasts reprogramming into iPSCs. We find that SIRT1 is required for efficient postreprogramming telomere elongation, and that this effect is mediated by a c-MYC-dependent regulation of the mTert gene. We further demonstrate that SIRT1-deficient iPSCs accumulate chromosomal aberrations and show a derepression of telomeric heterochromatin. Finally, SIRT1-deficient iPSCs form larger teratomas that are poorly differentiated, highlighting a role for SIRT1 in exit from pluripotency. In summary, this work demonstrates a role for SIRT1 in the maintenance of pluripotency and modulation of differentiation. © 2014 The Authors.

Abengozar M.A.,Proteomics Unit | De Frutos S.,Autonomous University of Madrid | Ferreiro S.,Animal Facility | Soriano J.,Confocal Microscopy Unit | And 8 more authors.
Blood | Year: 2012

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptorexpressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor-independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2- blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases. © 2012 by The American Society of Hematology.

Fernandez-Miranda G.,Cell Division and Cancer Group | Fernandez-Miranda G.,Barcelona Institute for Research in Biomedicine | Trakala M.,Cell Division and Cancer Group | Martin J.,Transgenic Mice Unit | And 7 more authors.
Development | Year: 2011

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, we show here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint. © 2011. Published by The Company of Biologists Ltd.

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