RESUMO
Here we report that DNA decatenation is not a physical requirement for the formation of mammalian chromosomes containing a two-armed chromosome scaffold. 2-aminopurine override of G2 arrest imposed by VM-26 or ICRF-193, which inhibit topoisomerase II (topo II)-dependent DNA decatenation, results in the activation of p34cdc2 kinase and entry into mitosis. After override of a VM-26-dependent checkpoint, morphologically normal compact chromosomes form with paired axial cores containing topo II and ScII. Despite its capacity to form chromosomes of normal appearance, the chromatin remains covalently complexed with topo II at continuous levels during G2 arrest with VM-26. Override of an ICRF-193 block, which inhibits topo II-dependent decatenation at an earlier step than VM-26, also generates chromosomes with two distinct, but elongated, parallel arms containing topo II and ScII. These data demonstrate that DNA decatenation is required to pass a G2 checkpoint, but not to restructure chromatin for chromosome formation. We propose that the chromosome core structure is templated during interphase, before DNA decatenation, and that condensation of the two-armed chromosome scaffold can therefore occur independently of the formation of two intact and separate DNA helices.
Assuntos
Proteínas Aviárias , Cromossomos/metabolismo , DNA/metabolismo , Fase G2/fisiologia , 2-Aminopurina/farmacologia , Animais , Antimetabólitos/farmacologia , Células CHO , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Cricetinae , DNA Topoisomerases Tipo II/fisiologia , Proteínas de Ligação a DNA/metabolismo , Dicetopiperazinas , Inibidores Enzimáticos/farmacologia , Rim , Mitose , Modelos Genéticos , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Teniposídeo/farmacologia , Inibidores da Topoisomerase IIRESUMO
Survivin, a dimeric baculovirus inhibitor of apoptosis repeat (BIR) motif protein that is principally expressed in G2 and mitosis, has been associated with protection against apoptosis of cells that exit mitosis aberrantly. Mammalian survivin has been reported to associate with centrosomes and with the mitotic spindle. We have expressed a human hemagglutinin-tagged survivin plasmid to determine its localization, and find instead that it clearly acts as a passenger protein. In HeLa cells, survivin first associates with the kinetochores, and then translocates to the spindle midzone during anaphase and, finally, to the midbody during cell cleavage. Its localization is similar to that of TD-60, a known passenger protein. Both a point mutation in the baculovirus IAP repeat motif (C84A) and a COOH-terminal deletion mutant (Delta106) of survivin fail to localize to either kinetochores or midbodies, but neither interferes with cell cleavage. The interphase localization of survivin is cell cycle regulated since in permanently transfected NIH3T3 cells it is excluded from the nuclei until G2, where it localizes with centromeres. Survivin remains associated with mitotic kinetochores when microtubule assembly is disrupted and its localization is thus independent of microtubules. We conclude that human survivin is positioned to have an important function in the mechanism of cell cleavage.
Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas/metabolismo , Células 3T3 , Motivos de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Divisão Celular , Imunofluorescência , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Mutação/genética , Proteínas de Neoplasias , Ligação Proteica , Transporte Proteico , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fuso Acromático/metabolismo , Survivina , Dedos de ZincoRESUMO
Taxol (paclitaxel) induces a microtubule hyperassembled state, and effectively blocks cells in mitosis. Here we report that Taxol also induces a stable late-G1 block in nontransformed REF-52 and WI-38 mammalian fibroblast cells, but not in T antigen-transformed cells of the same parental lineage. G1 arrest is characterized by partially dephosphorylated pRb, and inactive cdk2 kinase. Nontransformed cells recover normally from Taxol arrest. In contrast, T antigen transformed cells continue inappropriately past both G1 and G2-M in the presence of Taxol, and undergo a rapid death upon release. These results demonstrate a microtubule sensitive step in G1 regulation of nontransformed fibroblast cells. Also, Taxol selectively induces death of transformed cells, possibly because they slip the Taxol-dependent G1 arrest, as well as G2/M arrest, which are both specific to nontransformed cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Fibroblastos/citologia , Fase G1/efeitos dos fármacos , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/análise , Inibidores Enzimáticos , Humanos , Hidroxiureia/farmacologia , Proteínas Associadas aos Microtúbulos/análise , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Proteínas Serina-Treonina Quinases/análise , Ratos , Proteína do Retinoblastoma/análise , Vírus 40 dos Símios/imunologiaRESUMO
Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, alpha, gamma1, and delta, have nearly identical catalytic domains, but they vary in sequence at their extreme NH2 and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 alpha associates with the nuclear matrix, PP-1 gamma1 concentrates in nucleoli in association with RNA, and PP-1 delta localizes to nonnucleolar whole chromatin. During mitosis, PP-1 alpha is localized to the centrosome, PP-1 gamma1 is associated with microtubules of the mitotic spindle, and PP-1 delta strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.
Assuntos
Isoenzimas/análise , Mitose , Fosfoproteínas Fosfatases/análise , Sequência de Aminoácidos , Especificidade de Anticorpos , Células HeLa , Humanos , Interfase , Isoenzimas/imunologia , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/imunologia , Proteína Fosfatase 1 , Frações SubcelularesRESUMO
A "spindle assembly" checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21(WAF1) and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Divisão Celular , Citocalasina B/farmacologia , Fase G1 , Poliploidia , Fuso Acromático/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Separação Celular , Cromossomos/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Citocalasina B/análogos & derivados , Inibidores Enzimáticos/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Tubulina (Proteína)/metabolismoRESUMO
p21WAF1 and 14-3-3sigma, which are both transcriptional products of p53, have been reported to play a role in the G2 DNA damage checkpoint in mammalian cells. Human colon carcinoma cells, isogenic except for the presence or absence of either p21WAF1 or 14-3-3sigma (T. A. Chan et al., Genes Dev., 14: 1584-1588, 2000), are useful models for analysis of the role of these proteins in checkpoint control. Here, we have examined mitotic behavior within a single cell cycle after DNA damage in these cell lines. Our results show that p21WAF1, but not 14-3-3sigma, imposes a significant G2 delay after DNA damage. After G2 delay, we found that all isogenic cells, including those competent for both p21WAF1 and 14-3-3sigma, adapt to the DNA damage checkpoint and progress into mitosis, where they undergo incomplete chromosome segregation and reenter G1 with a tetraploid DNA content. Strikingly, our results show that p21WAF1, but not 14-3-3sigma, activates a checkpoint in response to DNA damage that prevents continued cycling of the tetraploid cells that result from a mitotic catastrophe characterized by failure to complete cell division. These results demonstrate that a tetraploid DNA content is not a reliable criterion to establish that arrest occurs in G2. Also, the DNA damage checkpoint mediated by p53-dependent induction of p21WAF1 assures neither G2 arrest nor DNA repair sufficient to enable accurate chromosome segregation in human colon carcinoma cells. We conclude that p21WAF1, but not 14-3-3sigma, has a unique role in the induction of G1 arrest in tetraploid cells that results from mitotic catastrophe after DNA damage.
Assuntos
Biomarcadores Tumorais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Ciclinas/fisiologia , Dano ao DNA , Exonucleases , Fase G2/fisiologia , Proteínas de Neoplasias , Proteínas/fisiologia , Proteínas 14-3-3 , Antineoplásicos/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/efeitos da radiação , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Exorribonucleases , Fase G2/genética , Humanos , Microscopia Confocal , Mitose/efeitos dos fármacos , Mitose/fisiologia , Mitose/efeitos da radiação , Índice Mitótico , Nocodazol/farmacologia , Ploidias , Células Tumorais CultivadasRESUMO
Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here were port the crystal structure of the shortest isoform of HBXIP (91 aa long,â¼11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a super family of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended ß-sheet area formed by 10 anti-parallel ß-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is dimeric form in solution. The fit between the experimental small angle X-ray scattering curve and the back calculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas Associadas aos Microtúbulos/metabolismo , Oncogenes , Transativadores/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/metabolismo , Vírus da Hepatite B , Humanos , Proteínas Inibidoras de Apoptose , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Survivina , Proteínas Virais Reguladoras e AcessóriasRESUMO
Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.