Centrosome Number Research Articles

Abstract 5091: Aneuploidy is in stomatin overexpressed cells

Abstract A 31.5 kDa membrane associated protein which is highly abundant in the lipid rafts is stomatin. Human gene expression profile from UCSC Genome Bioinformatics databank indicates that stomatin is major in the peripheral blood, adipose tissue, muscle, and bone marrow. In the beginning, stomatin was thought to be involved in the disease OHSt (overhydrated hereditary stomatocytosis), and therefore named stomatin. However, later studies did not reveal mechanistic role of stomatin in mediating OHSt by evaluating stomatin gene deleted mice; the function of stomatin is therefore remained unclear. In this study, we found that overexpression of stomatin was able to induce the multinucleated cell formation. Our data also demonstrated that stomatin made in the cytoplasm could be transported outside of the cell and released to the culture medium. While adding the culture medium or condition medium to the cells, which did not contain exogenous stomatin, these cells were also triggered to undergo formation of multinucleated cells. Additionally, the stomatin induced multinucleated cells presented aneuploidy and abnormal centrosome number. Possible mechanisms underlying functions of stomatin that induce multinucleated cells are investigated. Note: This abstract was not presented at the meeting. Citation Format: Jui-Hao Lee, Chi-Hung Lin. Aneuploidy is in stomatin overexpressed cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5091. doi:10.1158/1538-7445.AM2014-5091

Abstract 5085: Inducing multipolarity of acentrosomal mitotic spindles as a novel tumor-specific targeting strategy revealed by the antimitotic effect of mdivi-1

Abstract Traditional antimitotic drugs for cancer chemotherapy often have undesired toxicity to healthy tissue, limiting clinical application. Developing tumor-specific antimitotic strategies is necessary to improve the efficacy of drugs in the selective eradication of tumor cells. In this study, we discovered mdivi-1 (mitochondrial division inhibitor-1), which was originally reported as an inhibitor of mitochondrial fission protein dynamin-related protein 1 (Drp1), specifically disrupts M phase cell cycle progression only in human tumor cells and not in non-transformed fibroblasts and epithelial cells. The antimitotic effect of mdivi-1 is Drp1-independent, as M phase abnormality induced by mdivi-1 was observed in both Drp1 wild-type and Drp1 knockout MEF cells. We also identified that the tumor transformation process required for the antimitotic effect of mdivi-1 is downstream of SV40 large T and small t antigens - but not hTERT-mediated immortalization. Mdivi-1 induces multipolar mitotic spindles in tumor cells regardless of centrosome number. Acentrosomal spindle poles, which do not contain bona-fide centrosome components γ-tubulin and centrin-2, were found to contribute to the spindle multipolarity. The formation of such aberrant mitotic spindles leads to gross genome instability and apoptotic cell death through Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Gene expression profiling comparing non-transformed and transformed cells revealed that the genes involved in assembly of acentrosomal spindles are highly up-regulated in tumor cells, thus providing the mechanism underlying the preferential formation of acentrosomal multipolar spindles in tumor cells compared to normal cells after mdivi-1 treatment. Taken together, our studies imply that mdivi-1 represents a novel class of quinazolinone compounds that function as acentrosomal spindle inducers (ASI). We suggest that promoting the assembly of multipolar mitotic spindles composed of acentrosomal poles could serve as a novel strategy in achieving a tumor-specific antimitotic effect. (Work was supported by P30CA047904, GM087798, UPCI and PA Dept of Health CURE program) Note: This abstract was not presented at the meeting. Citation Format: Jingnan Wang, Wei Qian, Masahiro Shuda, Jianfeng Li, Lucas Santana-Santos, Robert W. Sobol, Bennett Van Houten. Inducing multipolarity of acentrosomal mitotic spindles as a novel tumor-specific targeting strategy revealed by the antimitotic effect of mdivi-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5085. doi:10.1158/1538-7445.AM2014-5085

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence.

Localization of MLH3 at the centrosomes.

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.

E2F activators signal and maintain centrosome amplification in breast cancer cells.

Centrosomes ensure accurate chromosome segregation by directing spindle bipolarity. Loss of centrosome regulation results in centrosome amplification, multipolar mitosis and aneuploidy. Since centrosome amplification is common in premalignant lesions and breast tumors, it is proposed to play a central role in breast tumorigenesis, a hypothesis that remains to be tested. The coordination between the cell and centrosome cycles is of paramount importance to maintain normal centrosome numbers, and the E2Fs may be responsible for regulating these cycles. However, the role of E2F activators in centrosome amplification is unclear. Because E2Fs are deregulated in Her2(+) cells displaying centrosome amplification, we addressed whether they signal this abnormal process. Knockdown of E2F1 or E2F3 in Her2(+) cells decreased centrosome amplification without significantly affecting cell cycle progression, whereas the overexpression of E2F1, E2F2, or E2F3 increased centrosome amplification in MCF10A mammary epithelial cells. Our results revealed that E2Fs affect the expression of proteins, including Nek2 and Plk4, known to influence the cell/centrosome cycles and mitosis. Downregulation of E2F3 resulted in cell death and delays/blocks in cytokinesis, which was reversed by Nek2 overexpression. Nek2 overexpression enhanced centrosome amplification in Her2(+) breast cancer cells silenced for E2F3, revealing a role for the E2F activators in maintaining centrosome amplification in part through Nek2.

Beclin 1 and UVRAG confer protection from radiation-induced DNA damage and maintain centrosome stability in colorectal cancer cells.

Beclin 1 interacts with UV-irradiation-resistance-associated gene (UVRAG) to form core complexes that induce autophagy. While cells with defective autophagy are prone to genomic instability that contributes to tumorigenesis, it is unknown whether Beclin1 or UVRAG can regulate the DNA damage/repair response to cancer treatment in established tumor cells. We found that siRNA knockdown of Beclin 1 or UVRAG can increase radiation-induced DNA double strand breaks (DSBs), shown by pATM and γH2Ax, and promote colorectal cancer cell death. Furthermore, knockdown of Beclin 1, UVRAG or ATG5 increased the percentage of irradiated cells with nuclear foci expressing 53BP1, a marker of nonhomologous end joining but not RAD51 (homologous recombination), compared to control siRNA. Beclin 1 siRNA was shown to attenuate UVRAG expression. Cells with a UVRAG deletion mutant defective in Beclin 1 binding showed increased radiation-induced DSBs and cell death compared to cells with ectopic wild-type UVRAG. Knockdown of Beclin 1 or UVRAG, but not ATG5, resulted in a significant increase in centrosome number (γ-tubulin staining) in irradiated cells compared to control siRNA. Taken together, these data indicate that Beclin 1 and UVRAG confer protection against radiation-induced DNA DSBs and may maintain centrosome stability in established tumor cells.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

NUSAP1 influences the DNA damage response by controlling BRCA1 protein levels

NUSAP1 has been reported to function in mitotic spindle assembly, chromosome segregation, and regulation of cytokinesis. In this study, we find that NUSAP1 has hitherto unknown functions in the key BRCA1-regulated pathways of double strand DNA break repair and centrosome duplication. Both these pathways are important for maintenance of genomic stability, and any defects in these pathways can cause tumorigenesis. Depletion of NUSAP1 from cells led to the suppression of double strand DNA break repair via the homologous recombination and single-strand annealing pathways. The presence of NUSAP1 was also found to be important for the control of centrosome numbers. We have found evidence that NUSAP1 plays a role in these processes through regulation of BRCA1 protein levels, and BRCA1 overexpression from a plasmid mitigates the defective phenotypes seen upon NUSAP1 depletion. We found that after NUSAP1 depletion there is a decrease in BRCA1 recruitment to ionizing radiation-induced foci. Results from this study reveal a novel association between BRCA1 and NUSAP1 and suggests a mechanism whereby NUSAP1 is involved in carcinogenesis.

Use of the essential autophagy protein beclin 1 to regulate DNA damage response and predict response to chemoradiation in rectal cancer.

492 Background: Beclin 1 interacts with its cofactor UVRAG to promote autophagy, a cellular degradation process induced by cellular stress. While cells with defective autophagy are prone to genomic instability, it is unknown whether Beclin 1 and/or UVRAG can regulate DNA damage/repair. Methods: Colorectal cancer cell lines (HT-29, DLD1) with Beclin 1 or UVRAG siRNA were irradiated ± 5-FU. DNA damage (pATM, pH2Ax, 53BP1, RAD51), cell death (annexin V, caspase-3, clonogenic survival), and centrosome number (γ-tubulin staining), were determined. Beclin 1 protein expression was analyzed in pretreatment endoscopic biopsies from 96 rectal carcinoma patients who received radiation + 5-FU followed by surgery. Results: Beclin 1 or UVRAG knockdown was shown to enhance DNA double-strand breaks (DSBs) and cell death induced by radiation ± 5-FU. Suppression of either gene increased the percentage of irradiated cells with nuclear foci expressing 53BP1 (nonhomologous end joining), but not RAD51 (homologous recombination). Beclin 1 was shown to regulate UVRAG, and a UVRAG deletion mutant defective in Beclin 1 binding decreased protection against radiation-induced DSBs. Knockdown of Beclin 1 or UVRAG increased centrosome number in irradiated cells. While autophagy inhibition by ATG5 or LC3 siRNA enhanced radiation-induced DSBs, they did not alter centrosome number suggesting independence from autophagy. In rectal carcinomas, high vs low level Beclin 1 expression was detected in 56 (58.3%) and 40 (41.7%) tumors, respectively. Pre-treatment Beclin 1 was not associated with clinicopathological variables. However, high vs low Beclin 1 predicted a significantly reduced pathological response (macroscopic vs. microscopic or no residual tumor) to chemoradiation (high: 14.2% vs. low: 40%; p = 0.017). High vs low Beclin 1 was independently associated with poorer overall survival (HR, 4.30; 95% CI, 1.42-16.47; p = 0.009). Conclusions: Beclin 1 and UVRAG confer protection against radiation-induced DNA damage and maintain centrosome stability. The ability of Beclin 1 overexpression to confer resistance to chemoradiation, suggests its potential utility as a predictive biomarker in rectal cancer.

The role of centrosomes in cells and their potential contribution to carcinogenesis

The centrosomes are subcellular organelles composed of two centrioles surrounded by a pericentriolar material. In animal cells they are responsible for the organization of the interphase microtubule cytoskeleton including microtubule nucleation and elongation, their attachment and release. The centrosomes are also involved in the construction of the mitotic spindle and chromosome segregation. More than a century ago it was suggested that these structures might be involved in human diseases, including cancer. Cancer cells show a high frequency of centrosome aberrations, especially amplification. Centrosome defects may increase the incidence of multipolar mitoses that lead to chromosomal segregation abnormalities and aneuploidy, which is the predominant type of genomic instability found in human solid tumors. The number of these organelles in cells is strictly controlled and is dependent on the proper process of centrosome duplication. Multiple genes that are frequently found mutated in cancers encode proteins which participate in the regulation of centrosome duplication and the numeral integrity of centrosomes. In recent years there has been growing interest in the potential participation of centrosomes in the process of carcinogenesis, especially because centrosome abnormalities are observed in premalignant stages of cancer development. The common presence of abnormal centrosomes in cancer cells and the role these organelles play in the cells suggest that the factors controlling the number of centrosomes may be potential targets for cancer therapy.

Partial Hepatectomy in Acetylation-Deficient BubR1 Mice Corroborates that Chromosome Missegregation Initiates Tumorigenesis.

BackgroundAneuploidy has been suggested as one of the major causes of cancer from the time of Boveri. In support of this notion, many studies have shown that cancer cells exhibit aneuploidy. However, there are evidences that do not support the aneuploidy hypothesis. We have previously reported that the spindle assembly checkpoint protein BubR1 is acetylated in mitosis and that the acetylation of BubR1 is crucial for checkpoint maintenance and chromosome-spindle attachment. Mice heterozygous for acetylation-deficient BubR1 (K243R/+) spontaneously develop cancer with chromosome instability. As K243R/+ mice develop hepatocellular carcinoma, we set out to test if chromosome mis-segregation was the cause of their liver cancer.MethodsPrimary hepatocytes in the regenerating liver after partial hepatectomy (PH) were analyzed and compared for various mitotic parameters.ResultsPrimary hepatocytes isolated from K243R/+ mice after PH displayed a marked increase of chromosome misalignment, accompanied by an increase of micronuclei. In comparison, the number of nuclei per cell and the centrosome numbers were not different between wild-type and K243R/+ mice. Taken together, chromosome mis-segregation provokes tumorigenesis in mouse liver.ConclusionOur results corroborate that PH provides a reliable tool for assessing mitotic infidelity and cancer in mice.

Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells

NIMA-related kinase 2 (Nek2), a serine–threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.

An IKKα-Nucleophosmin Axis Utilizes Inflammatory Signaling to Promote Genome Integrity

The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms by which cells protect themselves from inflammatory signals are unknown. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied bygenomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, proinflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a mechanism in which an IKKα-NPM axis may use inflammatory signals to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.

Centrosomal Abnormalities Characterize Human and Rodent Cystic Cholangiocytes and Are Associated with Cdc25A Overexpression

Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A invitro and invivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth invitro and invivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.

Abstract B96: Design, synthesis and biological evaluation of a novel allosteric inhibitor of HSET that damages cancer cells with supernumerary centrosomes.

Abstract Almost a century ago Theodor Boveri suggested that tumor cells differ from normal cells in their high incidence of centrosome amplification. However, only recently have new therapeutic strategies been explored in an attempt to exploit these differences and the role of kinesins in mitosis. Intense interest in the field has led to development of KSP and CENP-E inhibitors that have been tested clinically as treatments for human cancer. Success has been limited because both motor proteins are essential to normal mitosis and inhibition leads to mitotic arrest and associated neutropenia toxicity in normal cells. In contrast HSET is essential for survival of cancer cells with centrosome amplification and has been shown to be dispensable in normal cells. Hence, HSET inhibition offers a unique opportunity to selectively damage malignant cells with supernumerary centrosomes without affecting normal cells. In keeping with these findings we report discovery of a novel allosteric inhibitor of HSET, CW069, that does not disrupt division in normal human fibroblast cells, or in MCF-7 cells with normal centrosome numbers. In fact, CW069 induces multipolar mitosis exclusively in cancer cells with extra centrosomes, causing apoptosis via catastrophic aneuploidy. The increased multipolar mitoses induced in N1E-115 cells by inhibitor CW069 recapitulates the phenotype described here, and by others, for siRNA depletion of HSET. The inhibitor also reduces cell growth and centrosome clustering in cancer cells with a lower incidence of centrosome amplification, including BT549 and MDA-MB-231 breast cancer cells. This is consistent with recent reports that depletion of HSET in DNA damage repair deficient cells may be lethal even to cancer cells with low-level centrosome amplification. Taken together, these data indicate that CW069 inhibition of HSET is not restricted to use in N1E-115 cells with high centrosome amplification, and could be broadly applicable to a range of human cancers. What is more, CW069 does not decrease the clonogenic capacity of primary adult human bone marrow cells, suggesting that it would not cause neutropenia toxicity in normal cells. It is anticipated that HSET inhibition could have a greater therapeutic margin than KSP or CENP-E inhibition, and, to the best of our knowledge we have described the first allosteric inhibitor of HSET that reduces centrosome clustering but does not induce the mitotic phenotypes associated with inhibition of KSP or CENP-E. This selectivity for HSET is consistent with our computational model, which indicates that the HSET loop 5 displays dynamic conformational selection for CW069 that cannot be achieved by closely related KSP. In summary, CW069 not only represents a substantial advance toward new cancer therapeutics, but also offers researchers a unique tool to unveil the full details of HSET function in mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B96. Citation Format: Ciorsdaidh A. Watts, Frances M. Richards, Andreas Bender, Peter J. Bond, Oliver Korb, Oliver Kern, Michelle Riddick, Paul Owen, Rebecca M. Myers, Jordan Raff, Fanni Gergely, Duncan I. Jodrell, Steven V. Ley. Design, synthesis and biological evaluation of a novel allosteric inhibitor of HSET that damages cancer cells with supernumerary centrosomes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B96.

LIS1 controls mitosis and mitotic spindle organization via the LIS1–NDEL1–dynein complex

Heterozygous LIS1 mutations are responsible for the human neuronal migration disorder lissencephaly. Mitotic functions of LIS1 have been suggested from many organisms throughout evolution. However, the cellular functions of LIS1 at distinct intracellular compartments such as the centrosome and the cell cortex have not been well defined especially during mitotic cell division. Here, we used detailed cellular approaches and time-lapse live cell imaging of mitosis from Lis1 mutant mouse embryonic fibroblasts to reveal critical roles of LIS1 in mitotic spindle regulation. We found that LIS1 is required for the tight control of chromosome congression and segregation to dictate kinetochore-microtubule (MT) interactions and anaphase progression. In addition, LIS1 is essential for the establishment of mitotic spindle pole integrity by maintaining normal centrosome number. Moreover, LIS1 plays crucial roles in mitotic spindle orientation by increasing the density of astral MT plus-end movements toward the cell cortex, which enhances cortical targeting of LIS1-dynein complex. Overexpression of NDEL1-dynein and MT stabilization rescues spindle orientation defects in Lis1 mutants, demonstrating that mouse LIS1 acts via the LIS1-NDEL1-dynein complex to regulate astral MT plus-ends dynamics and establish proper contacts of MTs with the cell cortex to ensure precise cell division.

Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts

BackgroundKrüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.MethodsTo determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4−/−), we transfected Klf4−/−MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4+/+) and untransfected or mock-transfected Klf4−/−MEFs.ResultsWe show that overexpression of Klf4 in Klf4−/−MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4−/−MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.ConclusionTaken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

PPP1R42, a PP1 binding protein, regulates centrosome dynamics in ARPE‐19 cells

The centrosome is the primary site for microtubule nucleation in cells and orchestrates reorganisation of the microtubule cytoskeleton during the cell cycle. The activities of the centrosome must be closely aligned with progression of the cell cycle; misregulation of centrosome separation and duplication is a hallmark of cancer. In a subset of cells, including the developing spermatid, the centrosome becomes specialised to form the basal body thereby supporting growth of the axoneme in morphogenesis of cilia and flagella, structures critical for signalling and motility. Mammalian spermatogenesis is an excellent model system to investigate the transformations in cellular architecture that accompany these changes including formation of the flagellum. We have previously identified a leucine-rich repeat protein (PPP1R42) that contains a protein phosphatase-1 binding site and translocates from the apical nucleus to the centrosome at the base of the flagellum during spermiogenesis. In this manuscript, we examine localisation and function of PPP1R42 in a ciliated epithelial cell model as a first step in understanding the role of this protein in centrosome function and flagellar formation. We demonstrate that PPP1R42 localises to the basal body in ARPE-19 retinal epithelial cells. Co-localisation and co-immunoprecipitation experiments further show that PPP1R42 interacts with γ-tubulin. Inhibition of PPP1R42 with small interfering RNAs causes accumulation of centrosomes indicating premature centrosome separation. Importantly, the activity of two signalling molecules that regulate centrosome separation, PP1 phosphatase and NEK2 kinase, changes when PPP1R42 is inhibited: PP1 activity is reduced with a corresponding increase in NEK2 activity. We have identified a role for the PP1-binding protein, PPP1R42, in centrosome separation in ciliated ARPE-19 cells. Our finding that inhibition of PPP1R42 expression increases the number of centrosomes per cell is consistent with our model that PPP1R42 is a positive regulator of PP1. PPP1R42 depletion reduces the activity of PP1 leading to activation of NEK2, the kinase responsible for phosphorylation of centrosomal linker proteins promoting centrosome separation. This work identifies a new molecule localised to the centrosome and basal body with a role in the complex signalling network responsible for controlling centrosome activities.

Mitotic errors, aneuploidy and micronuclei in Hodgkin lymphoma pathogenesis

The Reed-Sternberg (RS) cell is the driving force behind Hodgkin lymphoma (HL), a unique malignancy in which the rare RS cell creates an inflammatory microenvironment that recruits a reactive tumor infiltrate. Well-known oncogenic factors such as nuclear factor kappa B (NFκB) signaling and Epstein-Barr virus infection are linked to HL pathogenesis but do not adequately explain the RS cell’s key pathologic features of multi-nucleation, abnormalities of centrosome function and number and aneuploidy. Chromosomal instability is also considered a key pathway in the origin of the RS cell, though the molecular mechanisms have largely been a “black box.” We demonstrated that the midbody kelch domain protein KLHDC8B protects against mitotic errors, centrosomal amplification and chromosomal instability. Here we discuss how the new findings linking KLHDC8B to mitotic integrity and faithful chromosomal segregation are providing mechanistic explanations for the origin of the RS cell and the molecular pathogenesis of chromosomal instability in HL.