Centrosome amplification is a frequent phenomenon in malignancies and may facilitate tumorigenesis by promoting chromosomal instability. On the other hand, a centrosome inactivation checkpoint comprising centrosome amplification leading to elimination of cells by mitotic catastrophe has been described in response to DNA damage by ionizing radiation or cytostatic drugs. So far, the exact nature of DNA damage-induced centrosome amplification, which might be overduplication or fragmentation of existing centrosomes, has been controversial. To solve this controversy, we have established a method to distinguish between these two possibilities using A549 cells expressing photoconvertible CETN2-Dendra2. In response to various DNA-damaging treatments, centrosome amplification but not fragmentation was observed. Moreover, centrosome amplification was preceded by excessive formation of centrin-containing centriolar satellites, which were identified as de novo-generated atypical centrin dots staining positive for centriolar satellite markers but negative or only weakly positive for other established centrosomal markers, and which could be verified as centriolar satellites using immunogold electron microscopy. In line with this notion, disruption of dynein-mediated recruitment of centrosomal proteins via centriolar satellites suppressed centrosome amplification after DNA damage, and excessive formation of centriolar satellites could be inhibited by interference with Chk1, a known mediator of centrosome amplification in response to DNA damage. In conclusion, we provide a model in which a Chk1-mediated DNA damage checkpoint induces excessive formation of centriolar satellites constituting assembly platforms for centrosomal proteins, which subsequently leads to centrosome amplification.

Centrosomes are central regulators of mitosis that are often amplified in cancer cells. Centrosomes function both as organizers of the mitotic spindle and as reaction centers to trigger activation of Cdk1 and G 2 /M transition in the cell cycle, but their functional organization remains incomplete. Recent proteomic studies have identified novel components of the human centrosome including Cep63, a protein of unknown function that Xenopus studies have implicated in mitotic spindle assembly and spindle inactivation after DNA damage. Here, we report that human Cep63 binds to and recruits Cdk1 to centrosomes, and thereby regulates mitotic entry. RNAi-mediated Cep63 depletion in U2OS cancer cells induced polyploidization through mitotic skipping. Elicitation of this phenotype was associated with downregulation of centrosomal Cdk1, mimicking the phenotype induced by direct depletion of Cdk1. In contrast, Cep63 overexpression induced de novo centrosome amplification during cell-cycle interphase. Induction of this phenotype was suppressible by cell treatment with the Cdk inhibitor roscovitine. In a survey of 244 neuroblastoma cases, Cep63 mRNA overexpression was associated with MYCN oncogene amplification and poor prognosis. In cultured cells, Cep63 overexpression was associated with an enhancement in replication-induced DNA breakage. Together, our findings define human Cep63 as a centrosomal recruitment factor for Cdk1 that is essential for mitotic entry, providing a physical link between the centrosome and the cell-cycle machinery. Cancer Res; 71(6); 2129-39. Ó2011 AACR.

Fragile sites are specific genomic loci that are especially prone to chromosome breakage. For the human genome there are 31 rare fragile sites and 88 common fragile sites listed in the National Center for Biotechnology Information database; however, the exact number remains unknown. In this study, unstable DNA sequences, which have been previously tagged with a marker gene, were cloned and provided starting points for the characterization of two aphidicolin inducible common fragile sites. Mapping of these unstable regions with six-color fluorescence in situ hybridization revealed two new fragile sites at 6p21 and 13q22, which encompass genomic regions of 9.3 and 3.1 Mb, respectively. According to the fragile site nomenclature they were consequently entitled as FRA6H and FRA13E. Both identified regions are known to be associated with recurrent aberrations in malignant and nonmalignant disorders. It is conceivable that these fragile sites result in genetic damage that might contribute to cancer phenotypes such as osteosarcoma, breast and prostate cancer. ' 2007 Wiley-Liss, Inc.Key words: fragile site; FRA6H; FRA13E; genomic instability; chromosome rearrangements Fragile sites are specific genomic loci that are especially prone to express genomic instability. They can be visualized as gaps and breaks on metaphase chromosomes after culturing cells under conditions of replication stress. Based on their incidence in the human population, they are divided into rare fragile sites, occurring in less than 5% of all individuals, and common fragile sites being a constitutive feature of the genome of probably all individuals. 1According to the National Center for Biotechnology Information (NCBI), there are 31 rare fragile sites and 88 common fragile sites in the human genome. However, the exact number of fragile sites remains unclear, since there are no stringent criteria for inclusion and there is no regular update of the database. 2The molecular basis for the expression of rare fragile sites is the dynamic mutation of expanding CGG trinucleotide or AT-rich minisatellite sequences that after reaching a certain threshold expansion account for the observed instability. [3][4][5][6] In contrast, the molecular basis for breakage of common fragile sites is still unclear. 12 However, analysis of the identified sequences did not reveal any particular sequence structure; an ATrichness and an enrichment of DNA flexibility structures seem to be the only shared features.13,14 It has been hypothesized that the AT-richness leads to an accumulation of DNA secondary structures, which might cause a delayed replication at fragile sites. [15][16][17][18] This perturbed replication at fragile sites was shown to activate cell cycle checkpoints in an ATR-dependent manner. 19 In line with this, several targets and modifiers of the ATR-pathway, such as BRCA1, SMC1, CHK1 and the Fanconi anemia pathway proteins, were reported to be involved in maintenance of fragile sites stability. [20][21][22][23] The replication perturbation may result in doubl...

Fragile sites are specific genomic loci that are particularly prone to chromosomal breakage. Based on their incidence in the human population, they are divided into rare fragile sites occurring in less than 5% of all individuals and common fragile sites being a constitutional feature of the genome of probably all individuals. In this study, cloning of unstable DNA sequences, which have been previously genetically tagged with a marker gene, was the basis for defining the genomic localization of the common fragile site FRA11G at 11q23.3. Mapping of the fragile site with six-color fluorescence in situ hybridization (FISH) resulted in the precise genomic localization of FRA11G to a 4.5 Mb region. The chromosomal subband 11q23.3 harbors both the common fragile site FRA11G and the rare fragile site FRA11B. Here, we show that FRA11G maps 0.8 Mb proximal to the genomic region previously defined to be affected by expression of FRA11B; thus, the common and the rare fragile sites at 11q23.3 encompass distinct genomic regions. The region of FRA11G is known to be involved in somatic and germline recurrent aberrations, and it is conceivable that genetic damage resulting from this fragile site might contribute to clinical phenotypes.

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Supplementary Video 1 from Cep63 Recruits Cdk1 to the Centrosome: Implications for Regulation of Mitotic Entry, Centrosome Amplification, and Genome Maintenance

<div>Abstract<p>Centrosomes are central regulators of mitosis that are often amplified in cancer cells. Centrosomes function both as organizers of the mitotic spindle and as reaction centers to trigger activation of Cdk1 and G₂/M transition in the cell cycle, but their functional organization remains incomplete. Recent proteomic studies have identified novel components of the human centrosome including Cep63, a protein of unknown function that <i>Xenopus</i> studies have implicated in mitotic spindle assembly and spindle inactivation after DNA damage. Here, we report that human Cep63 binds to and recruits Cdk1 to centrosomes, and thereby regulates mitotic entry. RNAi-mediated Cep63 depletion in U2OS cancer cells induced polyploidization through mitotic skipping. Elicitation of this phenotype was associated with downregulation of centrosomal Cdk1, mimicking the phenotype induced by direct depletion of Cdk1. In contrast, Cep63 overexpression induced <i>de novo</i> centrosome amplification during cell-cycle interphase. Induction of this phenotype was suppressible by cell treatment with the Cdk inhibitor roscovitine. In a survey of 244 neuroblastoma cases, Cep63 mRNA overexpression was associated with <i>MYCN</i> oncogene amplification and poor prognosis. In cultured cells, Cep63 overexpression was associated with an enhancement in replication-induced DNA breakage. Together, our findings define human Cep63 as a centrosomal recruitment factor for Cdk1 that is essential for mitotic entry, providing a physical link between the centrosome and the cell-cycle machinery. <i>Cancer Res; 71(6); 2129–39. ©2011 AACR.</i></p></div>

Supplementary Video 2 from Cep63 Recruits Cdk1 to the Centrosome: Implications for Regulation of Mitotic Entry, Centrosome Amplification, and Genome Maintenance