Centrosomal abnormality is common in and a potential biomarker for bladder cancer

Centrosomal abnormalities have been implicated in chromosomal segregation aberrations that result from the formation of multipolar mitotic spindles and lead to aneuploidy. Aneuploidy is a characteristic of neoplasia and underlies the development and progression of bladder cancer. Therefore, centrosomal abnormality may play a key role in urothelial tumor transformation. The purpose of our investigation was to determine whether centrosomal abnormalities are present in malignant urothelial cells, define the relationship between centrosomal abnormalities and aneuploidy and determine whether the presence of centrosomal abnormalities might be a potential diagnostic marker for bladder cancer. Bladder wash specimens obtained from patients with and without a history of urothelial carcinoma were analyzed for centrosomal abnormalities using an immunoassay with a gamma-tubulin antibody. FISH with centromeric probes for chromosomes 4 and 9 and DNA ploidy image analysis were performed to detect aneuploidy. Defective centrosomes were found in 40 of 45 bladder wash specimens from patients with bladder cancer but in none of the 10 samples from patients without it. A large percentage (69%) of grade 1 tumors were positive for centrosomal abnormalities, and these abnormalities were increasing in numbers and size in grade 2 (93%) and grade 3 (100%) specimens. Centrosomal abnormalities and numerical chromosomal aberrations frequently appeared concomitantly in the same malignant cells. All of the specimens showing aneuploidy also exhibited centrosomal abnormalities: centrosomal defects and aneuploidy occurred together in 80% of malignant bladder tumors, with an especially high percentage in higher-grade tumors. The overall positivity of centrosomal abnormalities was higher than that of aneuploidy (88% vs. 80%), especially in grade 1 tumors (69% vs. 46%), whereas aneuploidy was strongly associated with grade 2 and grade 3 tumors. Centrosomal abnormalities are common in bladder cancer, even in low-grade tumors, and strongly associated with cancer grade and aneuploidy, especially in high-grade neoplasms. Centrosomal abnormalities appear to be intrinsic to aneuploidy and tumorigenesis and may be potential markers for early detection of bladder cancer.     

Cep128 associates with Odf2 to form the subdistal appendage of the centriole

The mother centriole in a cell has two appendages, the distal appendage (DA) and subdistal appendage (SDA), which have roles in generating cilia and organizing the cellular microtubular network, respectively. In the knockout (KO) cells of Odf2, the component of the DA and SDA, both appendages simultaneously disappear. However, the molecular mechanisms by which the DA and SDA form independently but close to each other downstream of Odf2 are unknown. Here, using super‐resolution structured illumination microscopy (SR‐SIM), we found that the signal for GFP‐tagged Odf2 overlapped considerably with that of immunofluorescently labeled Cep128. We further found that Cep128 knockdown (KD) caused the dissociation of other SDA components from the centriole, including centriolin, Ndel1, ninein and Cep170, whereas Odf2 was still associated with the centriole. In contrast, the DA components remained associated with the centriole in Cep128 KD cells. Consistent with this observation, we identified Cep128 as an Odf2‐interacting protein by immunoprecipitation. Taken with the finding that Cep128 deletion decreased the stability of centriolar microtubules, our results indicate that Cep128 associates with Odf2 in the hierarchical assembly of SDA components to elicit the microtubule‐organizing function.     

Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells

Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.     

Microtubule organization during human parthenogenesis

In human fertilization, the sperm centrosome plays a crucial role as a microtubule organizing center (MTOC). We studied microtubule organization during human parthenogenesis, which occurs when a human egg undergoes cleavage without a sperm centrosome. Multiple cytoplasmic asters were organized in the human oocyte after parthenogenetic activation, indicating that multiple MTOC are present in the human oocyte cytoplasm and function like a human sperm centrosome during parthenogenesis.     

Oncogenic role of nuclear accumulated Aurora‐A

Aurora‐A, also known as Aik, BTAK, or STK15, is a centrosomal serine/threonine protein kinase, which is proto‐oncogenic and is overexpressed in a wide range of human cancers. Besides gene amplification and mRNA overexpression, proteolytic resistance mechanisms are thought to contribute to overexpression of Aurora‐A. However, it is not yet clear how overexpressed Aurora‐A affects the expression of transformed phenotype. Here, we found that nuclear accumulation of Aurora‐A was critical for transformation activity. Cellular protein fractionation experiments and immunoblot analysis demonstrated a predominance of Aurora‐A in the nuclear soluble fraction in head and neck cancer cells. Indirect immunofluorescence using confocal laser microscopy confirmed nuclear Aurora‐A in head and neck cancer cells, while most oral keratinocytes exhibited only centrosomal localization. The expression of nuclear export signal‐fused Aurora‐A demonstrated that the oncogenic transformation activity was lost on disruption of the nuclear localization. Thus, the cytoplasmic localization of overexpressed Aurora‐A previously demonstrated by immunohistochemical analysis is not likely to correspond to that in intact cancer cells. This study identifies an alternative mode of Aurora‐A overexpression in cancer, through nuclear rather than cytoplasmic functions. We suggest that substrates of Aurora‐A in the cell nuclear soluble fraction can represent a novel therapeutic target for cancer. © 2009 Wiley‐Liss, Inc.     

Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells

Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.     

Loss of γ-tubulin,GCP-WD/NEDD1 and CDK5RAP2 from the Centrosome of Neurons in Developing Mouse Cerebral and Cerebellar Cortex

It has been recently reported that the centrosome of neurons does not have microtubule nucleating activity. Microtubule nucleation requires γ-tubulin as well as its recruiting proteins, GCP-WD/NEDD1 and CDK5RAP2 that anchor γ-tubulin to the centrosome. Change in the localization of these proteins during in vivo development of brain, however, has not been well examined. In this study we investigate the localization of γ-tubulin, GCP-WD and CDK5RAP2 in developing cerebral and cerebellar cortex with immunofluorescence. We found that γ-tubulin and its recruiting proteins were localized at centrosomes of immature neurons, while they were lost at centrosomes in mature neurons. This indicated that the loss of microtubule nucleating activity at the centrosome of neurons is due to the loss of γ-tubulin-recruiting proteins from the centrosome. RT-PCR analysis revealed that these proteins are still expressed after birth, suggesting that they have a role in microtubule generation in cell body and dendrites of mature neurons. Microtubule regrowth experiments on cultured mature neurons showed that microtubules are nucleated not at the centrosome but within dendrites. These data indicated the translocation of microtubule-organizing activity from the centrosome to dendrites during maturation of neurons, which would explain the mixed polarity of microtubules in dendrites.     

Merlin/ERM proteins establish cortical asymmetry and centrosome position

The ability to generate asymmetry at the cell cortex underlies cell polarization and asymmetric cell division. Here we demonstrate a novel role for the tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin, and Moesin) in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of the actin regulator Ezrin, which in turn positions the interphase centrosome in single epithelial cells and three-dimensional organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. These data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex and suggest that Merlin''s role in restricting cortical Ezrin may contribute to tumorigenesis by disrupting cell polarity, spindle orientation, and, potentially, genome stability.