RESUMEN
The MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis. We have previously reported that endogenous levels of MdmX can suppress proliferation and promote pseudo-bipolar mitosis in primary and tumor cells derived from p53-deficient mice, and that MdmX-p53 double deficient mice succumb to spontaneously formed tumors more rapidly than p53-deficient mice. These results suggest that the MdmX oncoprotein may act as a tumor-suppressor in cancers with compromised p53 function. By using orthotopic transplantation and lung colonization assays in mice we now establish a p53-independent anti-oncogenic role for MdmX in tumor progression. We also demonstrate that the roles of MdmX in genome stability and in proliferation are two distinct functions encoded by the separate MdmX protein domains. The central Zn-finger domain suppresses multipolar mitosis and chromosome loss, whereas the carboxy-terminal RING domain suppresses proliferation of p53-deficient cells. Furthermore, we determine that it is the maintenance of genome stability that underlies MdmX role in suppression of tumorigenesis in hyperploid p53 mutant tumors. Our results offer a rationale for the increased metastatic potential of p53 mutant human cancers with aberrant MdmX function and provide a caveat for the application of anti-MdmX treatment of tumors with compromised p53 activity.
RESUMEN
Despite over one hundred years of research, the duplication of the centrosome is a poorly understood process. Three recent papers--exploring three different kinases--may have provided the answer.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Caenorhabditis elegans , Centrosoma/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Ciclo Celular/fisiología , Ciclina E/metabolismo , Ciclina E/fisiología , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/fisiología , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Región Organizadora del Nucléolo/metabolismo , Nucleofosmina , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor EphA1Asunto(s)
Ciclo Celular/fisiología , Centrosoma/fisiología , Replicación del ADN/fisiología , Animales , Polaridad Celular , Segregación Cromosómica/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Interfase/fisiología , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Nucleofosmina , Huso Acromático/fisiología , Ubiquitinas/metabolismoRESUMEN
Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing gamma-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures-distinct from elements of the microtubule-organizing center-that is required for the somatic cell cycle to progress through G1 into S phase. Once the cell is in S phase, these core structures are not needed for the G2-M phase transition.
Asunto(s)
Centrosoma/fisiología , Fase G1 , Fase S , Animales , Antígenos/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Centriolos/fisiología , Chlorocebus aethiops , Gránulos Citoplasmáticos/fisiología , Gránulos Citoplasmáticos/ultraestructura , Interfase , Microscopía por Video , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Mitosis , Paclitaxel/farmacología , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Tubulina (Proteína)/metabolismoAsunto(s)
Ciclo Celular/fisiología , Centrosoma/fisiología , Animales , Núcleo Celular/fisiología , HumanosRESUMEN
Reproductive cells that are destined to become sperm or egg undergo meiotic division during which the chromosome number is halved. As Sluder and McCollum explain in their Perspective, new findings (Shonn et al.) in yeast show that there is a spindle checkpoint that operates during meiosis to ensure that an equal number of replicated chromosomes arrives at each pole of the cell. One of the components of this meiotic spindle checkpoint turns out to be Mad2, which gives the signal to halt meiosis if it looks like unequal chromosome segregation is taking place.
Asunto(s)
Proteínas Portadoras , Meiosis/fisiología , Proteínas de Unión al Calcio/fisiología , Proteínas de Ciclo Celular , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Segregación Cromosómica , Proteínas Fúngicas/fisiología , Humanos , Proteínas Nucleares , Saccharomycetales/genética , Saccharomycetales/fisiología , Transducción de SeñalRESUMEN
Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/fisiología , Dictyostelium/fisiología , Endocitosis , Neuropéptidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Actinas/inmunología , Animales , Adhesión Celular , División Celular , Membrana Celular/metabolismo , Movimiento Celular , Células Cultivadas , Quimiotaxis , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Humanos , Mutagénesis Sitio-Dirigida , Neuropéptidos/genética , Proteínas Recombinantes de Fusión , Homología de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
The p34(cdc2) protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Calcio/metabolismo , Mitosis/fisiología , Proteína Quinasa C/metabolismo , Erizos de Mar/enzimología , Animales , Proteína Quinasa CDC2/fisiología , Carcinógenos/farmacología , Ciclo Celular/efectos de los fármacos , Ciclina B/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Activación Enzimática , Activadores de Enzimas/farmacología , Mitosis/efectos de los fármacos , Fosforilación , Erizos de Mar/embriología , Erizos de Mar/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
It is of great importance for the cell to precisely coordinate the doubling of the interphase centrosome with nuclear events during the cell cycle and limit the number of centrosomes it contains at the onset of mitosis to two and only two. The penalties for mistakes are abnormal spindle assembly, inappropriate chromosome distribution, and consequently, genomic instability. We review the functional properties of the mechanisms that control when the centrosome duplicates in the cell cycle and the controls for centrosome copy number. We look to limits that are intrinsic to the centrosome itself and controls imposed by cell cycle linked changes in cytoplasmic conditions. Control of centrosome reproduction is exercised at both levels.
Asunto(s)
Ciclo Celular/fisiología , Células/enzimología , Centrosoma/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Animales , Células/citologíaRESUMEN
In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.
Asunto(s)
Citoplasma/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Fosfatasas cdc25 , Animales , Afidicolina/farmacología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Fertilización/fisiología , Ficusina/farmacología , Masculino , Modelos Genéticos , Fosfoproteínas Fosfatasas/metabolismo , Erizos de Mar , Albúmina Sérica Bovina/metabolismo , Factores Sexuales , Factores de Tiempo , Aglutininas del Germen de Trigo/metabolismoRESUMEN
The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Centrosoma/metabolismo , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Proteínas Supresoras de Tumor , Animales , Afidicolina/farmacología , Blastómeros/química , Extractos Celulares , Centrosoma/química , Ciclina E/análisis , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Proteínas Asociadas a Microtúbulos/farmacología , Óvulo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas , Proteínas Recombinantes/farmacología , Xenopus , Proteínas de XenopusRESUMEN
Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1-cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Delta-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase-anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary "licensing" event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.
Asunto(s)
Núcleo Celular/fisiología , Centrosoma/fisiología , Fase S/fisiología , Cigoto/citología , Anafase/fisiología , Animales , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Centrosoma/ultraestructura , Femenino , Masculino , Metafase/fisiología , Microscopía Electrónica , Mitosis/fisiología , Profase/fisiología , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Reproducción/fisiología , Fase S/efectos de los fármacos , Erizos de Mar , Cigoto/enzimologíaRESUMEN
Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BDelta90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.
Asunto(s)
Anafase/fisiología , Proteína Quinasa CDC2/fisiología , Ciclinas/metabolismo , Huso Acromático/fisiología , Actinas/análisis , Animales , División Celular , Línea Celular , Ciclinas/genética , Riñón/citología , Metafase , Microinyecciones , Microtúbulos , Miosinas/análisis , ARN Mensajero , RatasRESUMEN
During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 microm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a "start anaphase" reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.
Asunto(s)
Ciclo Celular , Mitosis , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Animales , Fusión Celular , Línea Celular , Dipodomys , Cinetocoros/fisiología , Cinetocoros/ultraestructura , Metafase , Microscopía Fluorescente , Microscopía de Interferencia , Factores de Tiempo , VertebradosRESUMEN
Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a 'spindle assembly' checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.