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Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR. [ABSTRACT FROM AUTHOR]

Abstract: With regard to cancer development the centrosome has been the center of attraction of scientists for already more than a 100years. After the initial assumption that amplified centrosomes and abnormal mitotic arrangements might be a cause of cancer at the beginning of the last century, enormous efforts have been undertaken to clarify the relevance of centrosome amplification in tumorigenesis. In the meantime, centrosome amplification has been observed in most, both solid and hematological, cancer entities and by now is viewed as a “hallmark” of cancer cells. In this review we summarize basics in centrosome biology and what is known about the emergence of amplified centrosomes. In addition, we discuss how centrosome amplification might cause aneuploidy thereby leading to malignant transformation of cells. Furthermore, we present recent insights into the role of centrosome amplification in tumor formation based on work in model systems. [Copyright &y& Elsevier]

Abstract: Centrosome abnormalities occur commonly in cancer, and contribute to chromosomal instability and tumorigenesis. New evidence on a phylogenetically conserved mechanism termed ‘centrosomal clustering’ provides exciting insights into how cells with supernumerary centrosomes adapt to avoid lethal multipolar divisions. Here, we highlight the emerging molecular basis of centrosome clustering, and its impact on asymmetric divisions of stem cells, chromosomal (in)stability and malignant transformation. Finally, pharmacological inhibition of centrosome clustering promises to selectively target tumor cells. [Copyright &y& Elsevier]

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia. [ABSTRACT FROM AUTHOR]

Abstract: As the primary microtubule organizing center of most eukaryotic cells, centrosomes play a fundamental role in proper formation of the mitotic spindle and subsequent chromosome separation. Normally, the single centrosome of a G1 cell duplicates precisely once prior to mitosis in a process that is intimately linked to the cell division cycle via cyclin-dependent kinase (cdk) 2 activity that couples centrosome duplication to the onset of DNA replication at the G1/S transition. Accurate control of centrosome duplication is critical for symmetric mitotic spindle formation and thereby contributes to the maintenance of genome integrity. Numerical and structural centrosome abnormalities are hallmarks of almost all solid tumors and have been implicated in the generation of multipolar mitoses and chromosomal instability. In addition to solid neoplasias, centrosome aberrations have recently been described in several different hematological malignancies like acute myeloid leukemias, myelodysplastic syndromes, Hodgkin''s as well as non-Hodgkin''s lymphomas, chronic lymphocytic leukemias and multiple myelomas. In analogy to many solid tumors a correlation between centrosome abnormalities on the one hand and karyotype aberrations as well as clinical aggressiveness on the other hand seems to exist in myeloid malignancies, chronic lymphocytic leukemias and at least some types of non-Hodgkin''s lymphomas. Molecular mechanisms responsible for the development of centrosome aberrations are just beginning to be unraveled. In general, two models with distinct functional consequences can be envisioned. First, centrosome aberrations can arise as a consequence of abortive mitotic events and impaired cytokinesis. Second, evidence has been provided that centrosome amplification can also precede genomic instability and arise in normal, diploid cells. Accordingly, this review will focus on recent advances in the understanding of both, causes and consequences of centrosome aberrations in hematological malignancies. [Copyright &y& Elsevier]

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation. [ABSTRACT FROM AUTHOR]

Centrosomes, which mediate accurate chromosome segregation during mitosis, undergo duplication precisely once per cell division at the G1/S boundary. Recently, we described centrosome aberrations as a possible cause of aneuploidy in acute myeloid leukemia (AML) and found a correlation of the percentage of cells carrying abnormal centrosomes to their cytogenetic risk profile. To elucidate the molecular events responsible for the development of centrosome aberrations in AML, tumor RNA of 29 AML samples was hybridized to cDNA microarrays. The microarrays comprised some 2800 different genes with relevance to hematopoiesis, tumorigenesis and mitosis and included a set of 359 centrosome-associated genes. We identified two gene expression signatures, which allowed an accurate classification according to the extent of centrosome aberrations and the ploidy status in 28 of 29 patients each. Specifically, 18 genes were present in both signatures, including genes that code for cell cycle regulatory proteins (cyclin A2, cyclin D3, cyclin H, CDK6, p18INK4c, p21Cip1, PAK1) and centrosome-associated proteins (pericentrin, a2-tubulin, NUMA1, TUBGCP2, PRKAR2A). In conclusion, the high expression of centrosome-associated genes matches the description of centrosome aberrations in several tumor types. Moreover, in AML the identification of G1/S-phase stimulatory genes suggests that one mechanism of aneuploidy induction might be the deregulation of centrosome replication at the G1/S boundary.Oncogene (2004) 23, 2379-2384. doi:10.1038/sj.onc.1207401 Published online 9 February 2004 [ABSTRACT FROM AUTHOR]

Stil (Sil, SCL/TAL1 interrupting locus) is a cytosolic and centrosomal protein expressed in proliferating cells that is required for mouse and zebrafish neural development and is mutated in familial microcephaly. Recently the Drosophila melanogaster ortholog of Stil was found to be important for centriole duplication. Consistent with this finding, we report here that mouse embryonic fibroblasts lacking Stil are characterized by slow growth, low mitotic index and absence of clear centrosomes. We hypothesized that Stil regulates mitosis through the tumor suppressor Chfr, an E3 ligase that blocks mitotic entry in response to mitotic stress. Mouse fibroblasts lacking Stil by genomic or RNA interference approaches, as well as E9.5 Stil-/- embryos, express high levels of the Chfr protein and reduced levels of the Chfr substrate Plk1. Exogenous expression of Stil, knockdown of Chfr or overexpression of Plk1 reverse the abnormal mitotic phenotypes of fibroblasts lacking Stil. We further demonstrate that Stil increases Chfr auto-ubiquitination and reduces its protein stability. Thus, Stil is required for centrosome organization, entry into mitosis and cell proliferation, and these functions are at least partially mediated by Chfr and its targets. This is the first identification of a negative regulator of the Chfr mitotic checkpoint. [ABSTRACT FROM AUTHOR]

Summary: Timely and accurate assembly of the mitotic spindle is critical for the faithful segregation of chromosomes, and centrosome separation is a key step in this process. The timing of centrosome separation varies dramatically between cell types; however, the mechanisms responsible for these differences and its significance are unclear. Here, we show that activation of epidermal growth factor receptor (EGFR) signaling determines the timing of centrosome separation. Premature separation of centrosomes decreases the requirement for the major mitotic kinesin Eg5 for spindle assembly, accelerates mitosis, and decreases the rate of chromosome missegregation. Importantly, EGF stimulation impacts upon centrosome separation and mitotic progression to different degrees in different cell lines. Cells with high EGFR levels fail to arrest in mitosis upon Eg5 inhibition. This has important implications for cancer therapy because cells with high centrosomal response to EGF are more susceptible to combinatorial inhibition of EGFR and Eg5. [Copyright &y& Elsevier]

In contrast to normal cells, malignant cells are frequently aneuploid and contain multiple centrosomes. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles in many cancer cells. Recently, we have shown that griseofulvin forces tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. Here, we describe the characterization of the novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering inmalignant cells. At concentrations where GF-15 had no significant impact on tubulin polymerization, spindle tension was markedly reduced in mitotic cells upon exposure to GF-15. Moreover, isogenic cells with conditional centrosome amplification were more sensitive to GF-15 than parental controls. In a wide array of tumor cell lines, mean inhibitory concentrations (IC50) for proliferation and survival were in the range of 1 to 5 µmol/L and were associated with apoptotic cell death. Importantly, treatment of mouse xenograft models of human colon cancer and multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival. These results show the in vitro and in vivo antitumor efficacy of a prototype small molecule inhibitor of centrosomal clustering and strongly support the further evaluation of this new class of molecules. [ABSTRACT FROM AUTHOR]

Abstract: Centrosomes are cytoplasmic organelles playing a fundamental role in organizing both the interphase cytoskeleton and the bipolar mitotic spindle. In addition, the centrosome has recently come into focus as part of the network that integrates cell cycle arrest and repair signals in response to genotoxic stress—the DNA damage response. One important mediator of this response, the checkpoint kinase Chk1, has been shown to negatively regulate the G2/M transition via its centrosomal localization. Moreover, there is growing evidence that a centrosome inactivation checkpoint exists, which utilizes DNA damage-induced centrosome fragmentation or amplification to provoke a “mitotic catastrophe” and eliminate damaged cells. Candidate regulators of this centrosomal checkpoint include the checkpoint kinase Chk2 and its upstream regulators ATM and ATR. In addition, a growing number of other proteins have been implicated in centrosomal regulation of the DNA damage response, e.g. the tumor suppressor p53, the breast cancer susceptibility gene product BRCA1 and mitotic regulators such as Aurora A, Nek2 and the Polo-like kinases Plk1 and Plk3. However, many missing links and discrepancies between different model systems remain. [Copyright &y& Elsevier]

A letter to the editor is presented in response to the article "Cep63 Recruits Cdk1 to the Centrosome: Implications for Regulation of Mitotic Entry, Centrosome Amplification, and Genome Maintenance," by H. Löffler et al. in the 2011 issue.