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1.
Blood ; 129(18): 2479-2492, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28270450

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.


Assuntos
Proliferação de Células/fisiologia , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Ilhas de CpG/fisiologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Proteínas Supressoras da Sinalização de Citocina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
FASEB J ; 29(5): 1999-2009, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25667221

RESUMO

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/genética , Citocinese/fisiologia , Imunofluorescência , Heterozigoto , Homozigoto , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Proteínas de Peixe-Zebra/genética
3.
J Biol Chem ; 289(26): 18514-25, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24828503

RESUMO

FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas F-Box/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Fase G2 , Humanos , Ligação Proteica , Proteólise , Proteínas Supressoras de Tumor/genética , Ubiquitinação
4.
EMBO Mol Med ; 10(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30108112

RESUMO

The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers.


Assuntos
Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Citocinese , Mitose , Proteínas Nucleares/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Morte Celular , Linhagem Celular Tumoral , Ciclina B/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Proteínas Nucleares/genética
5.
Cell Cycle ; 12(6): 899-906, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23442802

RESUMO

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Interfase , Proteínas de Ancoragem à Quinase A , Autoantígenos , Proteínas de Ligação a Calmodulina , Proteínas de Ciclo Celular/deficiência , Linhagem Celular Tumoral , Núcleo Celular/fisiologia , Proteínas do Citoesqueleto , Células HeLa , Humanos , Interfase/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)
6.
Mol Cell Biol ; 33(6): 1210-22, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23319047

RESUMO

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.


Assuntos
Dano ao DNA , Replicação do DNA , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Aberrações Cromossômicas , Reparo do DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos/metabolismo , Expressão Gênica/genética , Células HEK293 , Histonas/genética , Histonas/metabolismo , Recombinação Homóloga/genética , Humanos , Hidroxiureia/metabolismo , Lamina Tipo A/metabolismo , Células MCF-7 , Camundongos , Radiação Ionizante , Transdução de Sinais/genética
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