Regulation Of Centrosome Duplication Research Articles

Mitotic Aberration Coupled With Centrosome Amplification Is Induced by Hepatitis B Virus X Oncoprotein via the Ras-Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase-Mitogen-Activated Protein Pathway

Abstract Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n ≥ 3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.

Stability of centrosome numbers in poly(ADP-ribose) polymerase-1- and poly(ADP-ribose) glycohydrolase-deficient mouse ES cells

Poly(ADP-ribose) polymerase-1 (Parp-1) localizes mainly in the nucleus and functions in DNA repair, genome stability and cell death regulation. Meanwhile, it also localizes in centrosomes and is involved in the regulation of centrosome duplication. An abnormal increase in centrosome numbers is frequently observed in Parp-1-deficient (Parp-1-/-) mouse embryonic fibroblasts (MEFs) (Kanai et al. (2003) Mol. Cell. Biol. 23, 2451-2462). However, there are no studies on whether the centrosome abnormality occurs also in other cell types under Parp-1 deficiency. In this study, we report that Parp-1-/- mouse embryonic stem (ES) cell lines did not show an abnormally increased number of centrosomes compared to wild-type ES cells. Recently, poly(ADP-ribose) glycohydrolase (Parg) has also been shown to localize in centrosomes (Ohashi et al. (2003) Biochem. Biophys. Res. Commun. 307, 915-921). The number of centrosomes of Parg-deficient (Parg-/-) ES cells was also analyzed in this study and was found to be stable under Parg deficiency. We also examined centrosome numbers in wild-type, Parp-1-/- and Parg-/- ES cell lines after treatment with methylmethanesulfonate (MMS) or γ-irradiation. Although a slight increase in the number of centrosomes is observed in each genotype twenty-four hours after treatment with MMS at 50 μM or with γ-irradiation at 1.4 Gy, there was no difference among the genotypes. These results suggest that loss of Parp-1 and Parg is insufficient to induce abnormality in centrosome numbers in ES cells and that ES cells possibly possess a strict mechanism for the maintenance of a normal number of centrosomes. (Contributed by Takashi SUGIMURA, M.J.A.)

Involvement of Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribosyl)ation in Regulation of Centrosome Function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

Roles of BRCA1 in centrosome duplication.

Centrosome duplication is under precise control and occurs only once in a normal mammalian cell cycle. Disruption of this process causes centrosome amplification, unequal segregation of chromosomes and, ultimately, tumorigenesis. Recent studies indicate that breast cancer suppressor gene 1 (BRCA1) plays an important role in regulating centrosome duplication. BRCA1 is located in the centrosome and binds to gamma-tubulin. It interacts with a variety of proteins that regulate centrosome duplication, including BRCA2, CDK2-Cyclin A, CDK2-Cyclin E, Gadd45, p21, p53 and Rb. Furthermore, targeted disruption of murine BRCA1 results in centrosome amplification, suggesting that BRCA1 serves as a negative regulator for centrosome duplication. This review will examine these data and discuss possible relationships between BRCA1 and its interacting proteins in centrosome duplication.

CP110, a Cell Cycle-Dependent CDK Substrate, Regulates Centrosome Duplication in Human Cells

Centrosome duplication and separation are linked inextricably to certain cell cycle events, in particular activation of cyclin-dependent kinases (CDKs). However, relatively few CDK targets driving these events have been uncovered. Here, we have performed a screen for CDK substrates and have isolated a target, CP110, which is phosphorylated by CDKs in vitro and in vivo. Human CP110 localizes to centrosomes. Its expression is strongly induced at the G1-to-S phase transition, coincident with the initiation of centrosome duplication. RNAi-mediated depletion of CP110 indicates that this protein plays an essential role in centrosome duplication. Long-term disruption of CP110 phosphorylation leads to unscheduled centrosome separation and overt polyploidy. Our data suggest that CP110 is a physiological centrosomal CDK target that promotes centrosome duplication, and its deregulation may contribute to genomic instability.

A Mammalian In Vitro Centriole Duplication System: Evidence for Involvement of CDK2/Cyclin E and Nucleophosmin/B23 in Centrosome Duplication

Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centrosomal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.

Difference in the centrosome duplication regulatory activity among p53 'hot spot' mutants: potential role of Ser 315 phosphorylation-dependent centrosome binding of p53.

The p53 tumor suppressor protein regulates centrosome duplication through multiple pathways, and p21(Waf1/Cip1) (Waf1), a major target of p53's transactivation function, has been shown to be one of the effectors. However, it had been unclear whether the p53's Waf1-independent centrosome duplication regulatory pathways require its transactivation function. In human cancers, specific residues of p53 are mutated at a high frequency. These 'hot spot' mutations abrogate p53's transactivation function. If p53 regulates centrosome duplication in a transactivation-independent manner, different 'hot spot' mutants may regulate centrosome duplication differently. To test this, we examined the effect of two 'hot spot' mutants (R175H and R249S) for their centrosome duplication regulatory activities. We found that R175H lost the ability to regulate centrosome duplication, while R249S partially retained it. Moreover, R249S associates with both unduplicated and duplicated centrosomes similar to wild-type p53, while R175H only associates with duplicated, but not unduplicated centrosomes. Since cyclin-dependent kinase 2 (CDK2) triggers initiation of centrosome duplication, and p53 is phosphorylated on Ser 315 by CDK2, we examined the p53 mutants with a replacement of Ser 315 to Ala (A) and Asp (D), both of which retain the transactivation function. We found that S315D retained a complete centrosome duplication activity, while S315A only partially retained it. Moreover, S315D associates with both unduplicated and duplicated centrosomes, while S315A associates with only duplicated, but not unduplicated centrosomes. Thus, p53 controls the centrosome duplication cycle both in transactivation-dependent and transactivation-independent manners, and the ability to bind to unduplicated centrosomes, which is controlled by phosphorylation on Ser 315, may be important for the overall p53-mediated regulation of centrosome duplication.

Re-evaluating centrosome function

Over the past 100 years, the centrosome has risen in status from an enigmatic organelle, located at the focus of microtubules, to a key player in cell-cycle progression and cellular control. A growing body of evidence indicates that centrosomes might not be essential for spindle assembly, whereas recent data indicate that they might be important for initiating S phase and completing cytokinesis. Molecules that regulate centrosome duplication have been identified, and the expanding list of intriguing centrosome-anchored activities, the functions of which have yet to be determined, promises continued discovery.

The Mouse Mps1p-like Kinase Regulates Centrosome Duplication

The yeast Mps1p protein kinase acts in centrosome duplication and the spindle assembly checkpoint. We demonstrate here that a mouse Mps1p ortholog (esk, which we designate mMps1p) regulates centrosome duplication. Endogenous mMps1p and overexpressed GFP-mMps1p localize to centrosomes and kinetochores in mouse cells. Overexpression of GFP-mMps1p causes reduplication of centrosomes during S phase arrest. In contrast, a kinase-deficient mutant blocks centrosome duplication altogether. Control of centrosome duplication by mMps1p requires a known regulator of the process, Cdk2. Inhibition of Cdk2 prevents centrosome reduplication and destabilizes mMps1p, causing its subsequent loss from centrosomes, suggesting that Cdk2 promotes mMps1p's centrosome duplication function by regulating its stability during S phase. Thus, mMps1p, an in vitro Cdk2 substrate, regulates centrosome duplication jointly with Cdk2.

Tumorigenesis as a consequence of genetic instability in Brca1 mutant mice

Germline mutations in Brca1 are responsible for most cases of familial breast and ovarian cancers, but somatic mutations in the gene are rarely detected in sporadic tumors. Moreover, mouse embryos carrying Brca1-null mutations or homozygous deletions of Brca1 exon 11 of ( Brca1 Δ11 /Δ11 ) die during gestation due to proliferation defects, raising questions about the mechanisms by which Brca1 represses tumor formation. Molecular analysis reveals that these Brca1 mutations cause hypersensitivity to γ-irradiation and chromosomal abnormalities in embryos and embryonic fibroblast cells (MEFs). Notably, Brca1 Δ11 /Δ11 MEFs maintain an intact G 1-S checkpoint, but are defective in G 2-M checkpoint control. They also contain multiple, functional centrosomes, which lead to unequal chromosome segregation and aneuploidy. These data uncover an essential role for Brca1 in maintaining genetic stability through regulation of centrosome duplication and G 2-M checkpoint, and provide a molecular basis for its role in tumorigenesis. Finally, we show that conditional mutation of Brca1 in mammary epithelium causes increased apoptosis and abnormal ductal development. Mammary tumor formation in mutant mice occurs after long latency and is associated with p53 mutations. These results are consistent with a model that Brca1 acts as a caretaker gene, whose absence does not directly initiate tumorigenesis, instead, causes genetic instability, which triggers further alterations and ultimately leads to tumor formation.

Centrosome Amplification and a Defective G 2–M Cell Cycle Checkpoint Induce Genetic Instability in BRCA1 Exon 11 Isoform–Deficient Cells

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G 1–S cell cycle checkpoint and proliferate poorly. However, a defective G 2–M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G 2–M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.

Amorphous no longer: the centrosome comes into focus

Recent genetic and biochemical studies have provided new insights into the molecular basis of centrosome-mediated microtubule nucleation. In addition, molecules and mechanisms involved in microtubule severing and stabilization at the centrosome, assembly of proteins onto centrosomes and regulation of centrosome duplication and separation are being defined. Characterization of centrosome function, together with studies implicating centrosomes in tumorigenesis and demonstrating that centrosomes are highly organized, are beginning to bring into focus an organelle once viewed as an ‘amorphous cloud’.

Centrosome amplification as a possible mechanism for numerical chromosome aberrations in cerebral primitive neuroectodermal tumors with TP53 mutations

Although alterations in chromosome number have frequently been detected in human tumor cells and associated with tumor initiation and progression, the causal mechanisms are still not understood. One protein known to be involved in maintaining genetic stability is tumor suppressor p53. In mice, p53 has been implicated in the maintenance of diploidy (Cross et al., 1995) and the regulation of centrosome duplication (Fukasawa et al., 1996). Here we report on cerebral primitive neuroectodermal tumors that lacked the wild-type p53 gene (TP53) and showed multiple numerical chromosome aberrations, as detected by comparative genomic hybridization. In these tumors, the centrosome number was significantly higher than in a control tumor without a detected TP53 mutation and with few chromosomal imbalances. These findings indicate that abnormal centrosome amplification can occur in human tumors lacking wild-type TP53 and may be a mechanism by which numerical chromosome aberrations are generated.

Abnormal centrosome amplification in the absence of p53.

The centrosome plays a vital role in mitotic fidelity, ensuring establishment of bipolar spindles and balanced chromosome segregation. Centrosome duplication occurs only once during the cell cycle and is therefore highly regulated. Here, it is shown that in mouse embryonic fibroblasts (MEFs) lacking the p53 tumor suppressor protein, multiple copies of functionally competent centrosomes are generated during a single cell cycle. In contrast, MEFs prepared from normal mice or mice deficient in the retinoblastoma tumor suppressor gene product do not display these abnormalities. The abnormally amplified centrosomes profoundly affect mitotic fidelity, resulting in unequal segregation of chromosomes. These observations implicate p53 in the regulation of centrosome duplication and suggest one possible mechanism by which the loss of p53 may cause genetic instability.