ATM is activated by mitotic stress and suppresses centrosome amplification in primary but not in tumor cells

Centrosome amplification has been proposed to contribute to the development of aneuploidy and genome instability. Here, we show that Ataxia-Telangiectasia Mutated (ATM) is localized to the centrosome and co-purified with gamma-tubulin. The importance of ATM in centrosome duplication is demonstrated in Atm-deficient primary mouse embryonic fibroblasts that display centrosome amplification. Interestingly, centrosome amplification was not observed in tumor cell lines derived from Atm and p21 double deficient mouse. Our results also indicate that both p53 and p21 operate in the same pathway as ATM in regulating centrosome biogenesis. Finally, a potential role of ATM in spindle checkpoint regulation is demonstrated by which ATM protein is activated by mitotic stress. These results suggest a role of ATM in spindle checkpoint regulation and indicate that ATM suppresses genome instability and cellular transformation by regulating centrosome biogenesis.     

Pin1 regulates centrosome duplication, and its overexpression induces centrosome amplification, chromosome instability, and oncogenesis

Phosphorylation on Ser/Thr-Pro motifs is a major mechanism regulating many events involved in cell proliferation and transformation, including centrosome duplication, whose defects have been implicated in oncogenesis. Certain phosphorylated Ser/Thr-Pro motifs can exist in two distinct conformations whose conversion in certain proteins is catalyzed specifically by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and is important for the activation of multiple oncogenic pathways, and its deletion suppresses the ability of certain oncogenes to induce cancer in mice. However, little is known about the role of Pin1 in centrosome duplication and the significance of Pin1 overexpression in cancer development in vivo. Here we show that Pin1 overexpression correlates with centrosome amplification in human breast cancer tissues. Furthermore, Pin1 localizes to and copurifies with centrosomes in interphase but not mitotic cells. Moreover, Pin1 ablation in mouse embryonic fibroblasts drastically delays centrosome duplication without affecting DNA synthesis and Pin1 inhibition also suppresses centrosome amplification in S-arrested CHO cells. In contrast, overexpression of Pin1 drives centrosome duplication and accumulation, resulting in chromosome missegregation, aneuploidy, and transformation in nontransformed NIH 3T3 cells. More importantly, transgenic overexpression of Pin1 in mouse mammary glands also potently induces centrosome amplification, eventually leading to mammary hyperplasia and malignant mammary tumors with overamplified centrosomes. These results demonstrate for the first time that the phosphorylation-specific isomerase Pin1 regulates centrosome duplication and its deregulation can induce centrosome amplification, chromosome instability, and oncogenesis.     

Transgenic Mouse Line with Green-fluorescent Protein-labeled Centrin 2 allows Visualization of the Centrosome in Living Cells

The centrosome plays diverse roles throughout the cellular mitotic cycle and in post-mitotic cells. Analysis of centrosome position and dynamics in living murine cells has been limited due to a lack of adequate reporters and currently requires either cell fixation/immunostaining or transfection with centrosome reporters. Here we describe the generation and characterization of a transgenic mouse line that constitutively expresses green fluorescent protein-labeled Centrin-2 (GFP-CETN2). The phenotype of the mouse is indistinguishable from wild-type and it displays a single pair of fluorescent centrioles in cells of every organ and time point examined. This model will be helpful for visualizing the centrosome in multiple experimental conditions.     

Centrosome aberrations in hematological malignancies

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.     

A regulatory role of polycystin-1 on cystic fibrosis transmembrane conductance regulator plasma membrane expression.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by genetic mutations in either PKD1 or PKD2, the genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. ADPKD is characterized by the formation of multiple, progressive, fluid-filled renal cysts. To elucidate the mechanism of fluid secretion by ADPKD cysts, we examined the effect of PC-1 on the plasma membrane expression of cystic fibrosis transmembrane conductance regulator (CFTR), a key Cl(-) secretory protein. Five stably transfected MDCK lines were used in this study: two transfected with empty vector (control cells) and three expressing human PC-1 (PC-1 cells). The cAMP-induced endogenous short circuit currents (I(sc)) were smaller in PC-1 cells than in control cells. Compared to control cells, PC-1 cells transiently expressing pEGFP-CFTR showed significant reduction of whole cell cAMP-activated Cl(-) currents. Cell surface biotinylation experiments also indicated a reduction in surface expression of CFTR in PC-1 cells compared to control. Furthermore, studies using CHO cells transiently expressing PC-1 and CFTR suggest the importance of the PC-1 COOH-terminus in the observed reduction of CFTR plasma membrane expression. No differences in either endogeneous K(+) currents or P2Y receptor responses were observed between PC-1 and control cells, indicating the specificity of PC-1's action. These results indicate that PC-1 selectively maintains low cell surface expression of CFTR. Moreover, these findings suggest that the malfunction of PC-1 enhances plasma membrane expression of CFTR, thus causing abnormal Cl(-)secretion into the cyst lumen.     

Aneuploidy,centrosome activity and chromosome instability in cells deficient in homologous recombination repair

Chromosome instability and loss or gain of chromosomes are changes characteristic of many tumour cells and human disorders. However, the mechanism of these changes has not yet been fully determined. We have recently shown that hamster cell lines deficient in homologous recombination repair (HRR) genes XRCC2 and XRCC3 have an elevated frequency of aneuploidy compared with wild-type cells and mutant cells transfected with the appropriate human gene. In addition, XRCC2 and XRCC3 deficient hamster cell lines show a high frequency of multiple centrosomes and abnormal spindle formation. Cells deficient in HRR show a high frequency of both chromosome-type and chromatid-type aberrations, which could potentially lead to mis-segregation. The role of chromosome aberrations and other factors, including chromosome lagging, premature chromatid separation, and centrosome malfunctioning on chromosome mis-segregation in irs1 and irs1SF cells have been investigated. In particular, the linkage of DNA repair proteins with centrosomes suggests a key role for the centrosome in controlling cellular repair processes.     

Induction of robust de novo centrosome amplification, high-grade spindle multipolarity and metaphase catastrophe: a novel chemotherapeutic approach

Centrosome amplification (CA) and resultant chromosomal instability have long been associated with tumorigenesis. However, exacerbation of CA and relentless centrosome declustering engender robust spindle multipolarity (SM) during mitosis and may induce cell death. Recently, we demonstrated that a noscapinoid member, reduced bromonoscapine, (S)-3-(R)-9-bromo-5-(4,5-dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo-[4,5-g]isoquinoline (Red-Br-nos), induces reactive oxygen species (ROS)-mediated autophagy and caspase-independent death in prostate cancer PC-3 cells. Herein, we show that Red-Br-nos induces ROS-dependent DNA damage that resulted in high-grade CA and SM in PC-3 cells. Unlike doxorubicin, which causes double-stranded DNA breaks and chronic G2 arrest accompanied by ‘templated'' CA, Red-Br-nos-mediated DNA damage elicits de novo CA during a transient S/G2 stall, followed by checkpoint abrogation and mitotic entry to form aberrant mitotic figures with supernumerary spindle poles. Attenuation of multipolar phenotype in the presence of tiron, a ROS inhibitor, indicated that ROS-mediated DNA damage was partly responsible for driving CA and SM. Although a few cells (∼5%) yielded to aberrant cytokinesis following an ‘anaphase catastrophe'', most mitotically arrested cells (∼70%) succumbed to ‘metaphase catastrophe,'' which was caspase-independent. This report is the first documentation of rapid de novo centrosome formation in the presence of parent centrosome by a noscapinoid family member, which triggers death-inducing SM via a unique mechanism that distinguishes it from other ROS-inducers, conventional DNA-damaging agents, as well as other microtubule-binding drugs.     

ECRG2 disruption leads to centrosome amplification and spindle checkpoint defects contributing chromosome instability

Cancer cells contain an abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Abnormal amplification of centrosomes and defects of spindle assembly checkpoint are the major causes of chromosome instability in cancer cells. Here we present biochemical evidence to suggest a role of ECRG2, a novel tumor suppressor gene, in maintaining chromosome stability. ECRG2 localized to centrosomes during interphase and kinetochores during mitosis. Further analysis revealed that ECRG2 participates in centrosome amplification in a p53-dependent manner. Depletion of ECRG2 not only destabilized p53, down-regulated p21, and increased the cyclin E/CDK2 activity, thus initiating centrosome amplification, but also abolished the ability of p53 localize to centrosomes. Overexpression of ECRG2 restored the p53-dependent suppression of centrosome duplication. Furthermore, ECRG2-depleted cells show severely disrupted spindle phenotype but fail to maintain the mitotic arrest due to minimal BUBR1 protein levels. Taken together, our results indicate that ECRG2 is important for ensuring centrosome duplication, spindle assembly checkpoint, and accurate chromosome segregation, and its depletion may contribute to chromosome instability and aneuploidy in human cancers.     

GAA repeat instability in Friedreich ataxia YAC transgenic mice

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.     

A tentative classification of centrosome abnormalities in cancer

Centrosome anomalies are detected in virtually all human cancers. They have been implicated in multipolar mitoses, chromosome missegregation, and genomic instability. Despite extensive studies on the type and frequency of centrosome anomalies, a causative relationship between centrosome aberrations and chromosomal instability has been difficult to establish. For example, centrosome amplification can be present without associated chromosomal instability. In addition, not all cells appear to be permissive for centrosome-related mitotic defects suggesting that cellular mechanisms that limit the harmful effects of spindle malformation on genome integrity may exist. This review proposes to classify centrosome abnormalities in tumor cells into three groups based on their relevance to genomic instability: primary centrosome overduplication, transient centrosome accumulation, and permanent centrosome accumulation. Whereas the first two categories are associated with an increased risk of chromosomal missegregation, the latter category may not contribute to the propagation of genomic instability. Therefore, centrosome anomalies should not per se be viewed as a universal cause of chromosomal instability, rather, they need to be assessed in the cellular context in which they occur.     

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis

Centrosomes in mammalian cells have recently been implicated in cytokinesis; however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.     

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.     

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.     

Bub1 up-regulation and hyperphosphorylation promote malignant transformation in SV40 tag-induced transgenic mouse models

Rodents do not naturally develop prostate cancer. Currently, most widely used genetically engineered mouse prostate cancer models use SV40 T/tag oncogene. To understand the mechanism underlying prostate cancer development in transgenic and knock-in SV40 Tag mouse models, we did cDNA microarray analyses, comparing gene expression profiles of prostate cancer tissues from early-, late-, and advance-stage androgen-independent prostate cancers. Of the 67 genes that were up-regulated by > or = 10-fold, 40 are known to be required for chromosome stability. In particular, the spindle checkpoint component Bub1 was persistently up-regulated from early to advanced androgen-independent prostate cancer lesions. Significantly, Bub1, which is required for accurate chromosome segregation during mitosis, has recently been reported to bind SV40 Tag. Consistent with a spindle checkpoint defect, flow cytometry experiments indicate that advanced androgen-independent prostate cancer tumors exhibit aneuploidy, along with up-regulation of levels of both Bub1 mRNA and Bub1 protein or hyperphosphorylation. Importantly, up-regulation and hyperphosphorylation of Bub1 were also observed in established human prostate cancer cell lines and in clinical studies. Furthermore, analysis of human prostate cancer lines showed impaired spindle checkpoint function and endoreduplication following exposure to spindle toxins. Small interfering RNA-mediated repression of Bub1 in the human prostate cancer line PC-3 restrained cell proliferation, an effect mimicked by inhibition of mitogen-activated protein kinase, an upstream activator of Bub1. Thus, by perturbing Bub1 function, our observations suggest a new mechanism whereby the SV40 Tag oncoprotein promotes chromosomal instability and aneuploidy in transgenic mouse prostate cancer models. Whereas the exact details of this mechanism remain unclear, our novel findings raise the possibility of exploiting Bub1 as a new therapeutic target in the treatment of prostate cancer, the most common cancer in adult men in North America.     

Disappearance of polycystic kidney disease in revertant c-myc transgenic mice

Nineteen SBM transgenic mouse lines specifically expressing the c-myc protooncogene in renal epithelium have developed polycystic kidney disease (PKD). Transgene expression is completely penetrant, leading to death from renal failure. In the course of continuous breeding of eight transgenic lines, all lines underwent spontaneous transgene mutations characterized by partial deletion and probable rearrangement of the transgene insert. Revertant mice and their progeny have no evidence of renal disease. This constitutes the first report of spontaneous mutations occurring within transgene inserts. The high spontaneous mutation frequency of 10^-2 to 10^-3 greatly exceeds that of naturally occurring mutations and is probably favored by the transgene's multiple tandem insertion. These spontaneous mutations demonstrate that the intact transgene is necessary and sufficient to produce the SBM phenotype. Further, these results implicate deregulation of factor(s) governing epithelial cell proliferation in the pathogenesis of PKD in SBM mice.     

The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.     

Nephrocystin-1 Forms a Complex with Polycystin-1 via a Polyproline Motif/SH3 Domain Interaction and Regulates the Apoptotic Response in Mammals

Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.     

Overexpression of the insulin receptor inhibitor PC-1/ENPP1 induces insulin resistance and hyperglycemia

The ectoenzyme PC-1 is an insulin receptor inhibitor that is elevated in cells and tissues of humans with type 2 diabetes (T2D). We have recently shown that acute PC-1 overexpression in liver causes insulin resistance and glucose intolerance in mice (3), but the chronic effects of PC-1 overexpression on these functions are unknown. Herein we produced transgenic mice overexpressing the potent q allele of human PC-1 in muscle and liver. Compared with controls, these mice had 2- to 3-fold elevations of PC-1 content in liver and 5- to 10-fold elevations in muscle. In the fed state, the PC-1 animals had 100 mg/dl higher glucose levels and sixfold higher insulin levels compared with controls. During glucose tolerance tests, these PC-1 animals had peak glucose levels that were >150 mg/dl higher than controls. In vivo uptake of 2-deoxy-d-glucose in muscle during insulin infusion was decreased in the PC-1 animals. These in vivo data support the concept, therefore, that PC-1 plays a role in insulin resistance and hyperglycemia and suggest that animals with overexpression of human PC-1 in insulin-sensitive tissues may be important models to investigate insulin resistance.     

A high-resolution structure of the EF-hand domain of human polycystin-2

Autosomal dominant polycystic kidney disease (ADPKD) affects over 1:1000 of the worldwide population and is caused by mutations in two genes, PKD1 and PKD2. PKD2 encodes a 968-amino acid membrane spanning protein, Polycystin-2 (PC-2), which is a member of the TRP ion channel family. The C-terminal cytoplasmic tail contains an EF-hand motif followed by a short coiled-coil domain. We have determined the structure of the EF-hand region of PC-2 using NMR spectroscopy. The use of different boundaries, compared with those used in previous studies, have enabled us to determine a high resolution structure and show that the EF hand motif forms a standard calcium-binding pocket. The affinity of this pocket for calcium has been measured and mutants that both decrease and increase its affinity for the metal ion have been created.