HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.     

p16INK4a Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells

Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16^INK4a generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16^INK4a cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16^INK4a activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16^INK4a suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.     

p16INK4a Prevents Centrosome Dysfunction and Genomic Instability in Primary Cells

Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16^INK4a generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16^INK4a cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16^INK4a activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16^INK4a suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.     

Involvement of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles

Hepatitis B virus (HBV) includes an X gene (HBx gene) that plays a critical role in liver carcinogenesis. Because centrosome abnormalities are associated with genomic instability in most human cancer cells, we examined the effect of HBx on centrosomes. We found that HBx induced supernumerary centrosomes and multipolar spindles. This effect was independent of mutations in the p21 gene. Furthermore, the ability of HBV to induce supernumerary centrosomes was dependent on the presence of physiological HBx expression. We recently showed that HBx induces cytoplasmic sequestration of Crm1, a nuclear export receptor that binds to Ran GTPase, thereby inducing nuclear localization of NF-kappaB. Consistently, supernumerary centrosomes were observed in cells treated with a Crm1-specific inhibitor but not with an HBx mutant that lacked the ability to sequester Crm1 in the cytoplasm. Moreover, a fraction of Crm1 was found to be localized at the centrosomes. Immunocytochemical and ultrastructural examination of these supernumerary centrosomes revealed that inactivation of Crm1 was associated with abnormal centrioles. The presence of more than two centrosomes led to an increased frequency of defective mitoses and chromosome transmission errors. Based on this evidence, we suggest that Crm1 is actively involved in maintaining centrosome integrity and that HBx disrupts this process by inactivating Crm1 and thus contributes to HBV-mediated carcinogenesis.     

p53-Driven apoptosis limits centrosome amplification and genomic instability downstream of NPM1 phosphorylation

Chromosome loss or gain is associated with a large number of solid cancers, providing genomic plasticity and thus adaptability to cancer cells. Numerical centrosome abnormalities arising from centrosome over-duplication or failed cytokinesis are a recognized cause of aneuploidy. In higher eukaryotic cells, the centrosome duplicates only once per cell cycle to ensure the formation of a bipolar mitotic spindle that orchestrates the balanced distribution of the sister chromatids to the respective daughter cells. Here we delineate the events that allow abnormal centrosome duplication, resulting in mitotic errors and incorrect chromosome segregation in cells with sustained cyclin-dependent kinase (CDK) activity. We have identified NPM1 as a substrate for CDK6 activated by the Kaposi's sarcoma herpesvirus (KSHV) D-type cyclin and shown that p53-driven apoptosis occurs downstream of NPM1 phosphorylation as a checkpoint mechanism that prevents accumulation of cells with supernumerary centrosomes. Our findings provide evidence that abnormal chromosome segregation in KSHV-infected cells is a direct consequence of NPM1 phosphorylation and predict that genomic instability is an inevitable consequence of latent KSHV infection.     

The retroviral oncoprotein Tax targets the coiled-coil centrosomal protein TAX1BP2 to induce centrosome overduplication

Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.     

Excess centrosomes disrupt endothelial cell migration via centrosome scattering

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome–MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.     

Ionizing radiation induces delayed hyperrecombination in Mammalian cells

Exposure to ionizing radiation can result in delayed effects that can be detected in the progeny of an irradiated cell multiple generations after the initial exposure. These effects are described under the rubric of radiation-induced genomic instability and encompass multiple genotoxic endpoints. We have developed a green fluorescence protein (GFP)-based assay and demonstrated that ionizing radiation induces genomic instability in human RKO-derived cells and in human hamster hybrid GM10115 cells, manifested as increased homologous recombination (HR). Up to 10% of cells cultured after irradiation produce mixed GFP(+/-) colonies indicative of delayed HR or, in the case of RKO-derived cells, mutation and deletion. Consistent with prior studies, delayed chromosomal instability correlated with delayed reproductive cell death. In contrast, cells displaying delayed HR showed no evidence of delayed reproductive cell death, and there was no correlation between delayed chromosomal instability and delayed HR, indicating that these forms of genome instability arise by distinct mechanisms. Because delayed hyperrecombination can be induced at doses of ionizing radiation that are not associated with significantly reduced cell viability, these data may have important implications for assessment of radiation risk and understanding the mechanisms of radiation carcinogenesis.     

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis.     

Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication

Centrosome duplication is tightly controlled during faithful cell division, and unnecessary reduplication can lead to supernumerary centrosomes and multipolar spindles that are associated with most human cancer cells. In addition to nucleocytoplasmic transport, the Ran-Crm1 network is involved in regulating centrosome duplication to ensure the formation of a bipolar spindle. Here, we discover that nucleophosmin (NPM) may be a Ran-Crm1 substrate that controls centrosome duplication. NPM contains a functional nuclear export signal (NES) that is responsible for both its nucleocytoplasmic shuttling and its association with centrosomes, which are Ran-Crm1-dependent as they are sensitive to Crm1-specific nuclear export inhibition, either by leptomycin B (LMB) or by the expression of a Ran-binding protein, RanBP1. Notably, LMB treatment induces premature centrosome duplication in quiescent cells, which coincides with NPM dissociation from centrosomes. Moreover, deficiency of NPM by RNA interference results in supernumerary centrosomes, which can be reversed by reintroducing wild-type but not NES-mutated NPM. Mutation of a potential proline-dependent kinase phosphorylation site at residue 95, from threonine to aspartic acid (T95D) within the NES motif, abolishes NPM association and inhibition of centrosome duplication. Our results are consistent with the hypothesis that the Ran-Crm1 complex may promote a local enrichment of NPM on centrosomes, thereby preventing centrosome reduplication.     

TORC1-mediated protein synthesis regulates cilia size and function

The centrosome is the microtubule organizing center important for the establishment of the mitotic spindle in animal cells. In mitosis, cells normally contain two centrosomes, one for each pole of the bipolar spindle. If a cell acquires additional centrosomes, it has the potential to build a multi-polar spindle which could lead to catastrophic errors in chromosome segregation. Although such an event is unlikely to produce viable daughter cells, an increase in centrosome number has been shown to cause chromosome instability and produce anneuploid daughter cells ^1-3. Accordingly, supernumerary centrosomes have been found in a variety of human cancers and accumulation of additional centrosomes has been associated with the process of tumorigenesis ^{1, 4-9}. Despite the obvious importance of regulating centrosome number, relatively little is known about how centrosome duplication is regulated. Perhaps surprisingly, several recent studies, including three articles in this issue of Cell Cycle, implicate proteins involved in the regulation of chromosome cohesion in the maintenance of centrosome number during mitosis ^10-14. Here we will discuss these findings and what they may tell us about the regulation of centrosome number.     

Breaking the ties that bind: new advances in centrosome biology

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.     

Centrosome overduplication and mitotic instability in PKD2 transgenic lines

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.     

Opposite Roles for p38MAPK-Driven Responses and Reactive Oxygen Species in the Persistence and Resolution of Radiation-Induced Genomic Instability

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as ⁵⁶Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a threshold-like response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.     

Role of induced genetic instability in the mutagenic effects of chemicals and radiation

Recent studies have demonstrated that cells exposed to ionizing radiation or alkylating agents can develop prolonged genetic instability. Induced genetic instability is manisested in multiple ways, including delayed reproductive death, an increased rate of point mutations, and an increased rate of chromosome rearrangements. In many respects these changes are similar to the genetic instability associated with cancer and some human genetic diseases. Therefore, as with cancer cells, multiple mechanisms may be involved, some occuring in the early stages and some in the later stages. The high percentage of cells that develop induced genetic instability after exposure to stress, and the prolonged period over which the instability occurs, indicates that the instability is not in response to residual damage in the DNA or mutations in specific genes. Instead, changes affecting most of the exposed cells, such as epigenetic alterations in gene expression or chain reactions of chromosome rearrangements, are a more likely explanation. Learning more about the mechanisms involved in this process is essential for understanding the consequences of exposure of cells to ionizing radiation or alkylating agents.     

The importance of a single primary cilium

The centrosome is the main microtubule-organizing center in animal cells, and helps to influence the morphology of the microtubule cytoskeleton in interphase and mitosis. The centrosome also templates the assembly of the primary cilium, and together they serve as a nexus of cell signaling that provide cells with diverse organization, motility, and sensory functions. The majority of cells in the human body contain a solitary centrosome and cilium, and cells have evolved regulatory mechanisms to precisely control the numbers of these essential organelles. Defects in the structure and function of cilia lead to a variety of complex disease phenotypes termed ciliopathies, while dysregulation of centrosome number has long been proposed to induce genome instability and tumor formation. Here, we review recent findings that link centrosome amplification to changes in cilium number and signaling capacity, and discuss how supernumerary centrosomes may be an important aspect of a set of cilia-related disease phenotypes.     

Centriolar distal appendages activate the centrosome‐PIDDosome‐p53 signalling axis via ANKRD26

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase‐2), whose activation results in cleavage of p53’s key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome‐dependent Caspase‐2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53‐dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26‐dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.     

The death-inducing effect and genomic instability

Exposure to ionizing radiation can induce a heritable change in the unirradiated progeny of irradiated cells. This non-targeted effect of ionizing radiation manifests as genomic instability, and although there is some debate as to the role of genomic instability in the carcinogenic process, it is thought by some to be an early step in radiation carcinogenesis. Although the mechanism of induction of genomic instability is not clearly understood, evidence suggests that secreted factors from irradiated cells may be involved. We have previously identified another non-targeted effect of ionizing radiation, the death-inducing effect. Exposure of unirradiated GM10115 cells to medium from chromosomally unstable clones was generally found to be cytotoxic. However, occasionally cells will survive in medium from unstable clones and can be clonally expanded. The absolute yield of survivors is independent of the initial number of cells plated when cell densities reached 5,000 or more cells/dish. After cytogenetic analysis of the surviving colonies, we found chromosomal instability in three of 40 clones analyzed, while some clones exhibited increased micronucleus frequency and HPRT mutation frequency. These data suggest that our chromosomally unstable GM10115 cells secrete factors that are cytotoxic to the majority of stable, parental cells but are also capable of inducing a heritable change in some of the survivors that can manifest as delayed genomic instability. These results suggest a mechanism whereby instability can be perpetuated through the influences of potentially cytotoxic factors produced by genomically unstable clones.