Dephosphorylation of Plk1 occurs through PP2A-B55/ENSA/Greatwall pathway during mitotic DNA damage recovery

Recovery from DNA damage is critical for cell survival. However, serious damage cannot be repaired, leading to cell death for prevention of abnormal cell growth. Previously, we demonstrated that 4N-DNA accumulates via the initiation of an abnormal interphase without cytokinesis and that re-replication occurs during a prolonged recovery period in the presence of severe DNA damage in mitotic cells. Mitotic phosphorylated Plk1 is typically degraded during mitotic exit. However, Plk1 has unusually found to be dephosphorylated in mitotic slippage without cytokinesis during recovery from mitotic DNA damage. Here, we investigated how Plk1 dephosphorylation is established during recovery from mitotic DNA damage. Mitotic DNA damage activated ATM and Chk1/2 and repressed Cdk1 and Greatwall protein kinase, followed by PP2A activation through the dissociation of ENSA and PP2A-B55. Interaction between Plk1 and PP2A-B55α or PP2A-B55δ was strongly induced during recovery from mitotic DNA damage. Moreover, the depletion of PP2A-B55α and/or PP2A-B55δ by siRNA transfection led to the recovery of Plk1 phosphorylation and progression of the cell cycle into the G₁ phase. Therefore, to adapt to severe DNA damage, the activated Greatwall/ENSA signaling pathway was repressed by ATM/Chk1/2, even in mitotic cells. Activation of the PP2A-B55 holoenzyme complex induced the dephosphorylation of Plk1 and Cdk1, and finally, mitotic slippage occurred without normal chromosome segregation and cytokinesis.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

Role of microtubule organization in centrosome migration and mitotic spindle formation in PtK1 cells

Summary Quinacrine, an acridine derivative, has previously been shown to disrupt lateral associations between non-kinetochore microtubules (nkMTs) of opposite polarity in PtK₁ metaphase spindles such that the balance of spindle forces is significantly altered. We extended the analysis of the spatial relationship of spindle microtubules (MTs) in this study by using quinacrine to compare ATP-dependent requirements for early prometaphase centrosome separation and spindle formation. The route used for centrosome migration can take a variety of pathways in PtK₁ cells, depending on the location of the centrosomes at the time of nuclear envelope breakdown. Following quinacrine treatment centrosome separation decresased by 1.9 to 14.0 m depending on the pathway utilized. However, birefringence of the centrosomal region increased approximately 50% after quinacrine treatment. Quinacrine-treated mid-prometaphase cells, where chromosome attachment to MTs had occurred, showed a decrease in spindle length of approximately 6.0 m with only a slight increase in astral birefringence. Computer-generated reconstructions of quinacrine-treated prometaphase cells were used to confirm changes in MT reorganization. Early-prometaphase cells showed more astral MTs (aMTs) of varied length while mid-prometaphase cells showed only a few short aMTs. Late prometaphase cells again showed a large number of aMTs. Our results suggest that: (1) quinacrine treatment affects centrosome separation, (2) recruitment of nkMTs by kinetochores is quinacrine-sensitive, and (3) development of the prometaphase spindle is dependent on quinacrine-sensitive lateral interactions between nkMTs of opposite polarity. These data also suggest that lateral interactions between MTs formed during prometaphase are necessary for centrosome separation and normal spindle formation but not necessarily chromosome motion.Abbreviations aMT(s) astral microtubule(s) - DIC differential interference contrast - MT(s) microtubule(s) - kMT(s) kinetochore microtubule(s) - NEB nuclear envelope breakdown - nkMT(s) non-kinetochore microtubule(s)     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.     

Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling

Malachite green (MG) is a triphenyl methane dye used in various fields that demonstrates high toxicity to bacteria and mammalian cells. When bud stage zebrafish embryos were treated with MG at 125, 150, and 175 ppb for 14 h, the development of trunk including intersomitic vessels was inhibited in MG-treated flk-1-GFP transgenic embyos. MG clearly induced whole growth retardation. MG induced severe cell death in trunk intersomite region of zebrafish embryos and in human vascular endothelial cells in a dose-dependent manner. MG inhibited heart rates and cardiac looping. MG attenuated whole blood formation and inhibited vascular endothelial growth factor (VEGF)-induced receptor (R)-2 phosphorylation in vascular endothelial cells. In conclusion, MG significantly alters the cardiovascular development causing growth retardation in zebrafish through the blocking VEGFR-2 activation in early cardiovascular development. It suggests that MG may be an environmental toxic agent with the potential to induce embryonic cardiovascular defects in vertebrates.     

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.     

Dishevelled,a Wnt signalling component,is involved in mitotic progression in cooperation with Plk1

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo‐like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)‐KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT‐KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus‐ends and the SAC in Plk1‐dependent and ‐independent manners.     

抑制Plk1表达对食管癌细胞化疗敏感性的影响

研究食管癌细胞中Plk1表达被抑制后对化疗药物敏感性的影响,为进一步研究开发基于Plk1食管癌分子靶向治疗奠定基础。化学合成法合成特异性的短链Plk1siRNA,脂质体转染法将短链siRNA分别导入3代表性的食管癌细胞系;采用RT-PCR和蛋白质印迹法确认siRNA对Plk1表达的抑制效果;MTT分析观察抑制Plk1表达对食管癌细胞化疗药物敏感性的影响。半定量RT-PCR和蛋白质印迹分析结果表明,合成的特异性短链Plk1siRNA在mRNA和蛋白质水平上均能高效、特异性地抑制所使用的3代表性食管癌细胞系中Plk1的表达,抑制程度约90%。MTT分析结果表明,siRNA抑制Plk1表达后,能显著提高3食管癌细胞系对化疗药物阿霉素和顺铂的敏感性。由此证实,siRNA介导的Plk1表达抑制能显著提高食管癌细胞对化疗药物阿霉素和顺铂的敏感性。     

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells.     

Plk1 Protein Phosphorylates Phosphatase and Tensin Homolog (PTEN) and Regulates Its Mitotic Activity during the Cell Cycle

PTEN is a well known tumor suppressor through the negative regulation of the PI3K signaling pathway. Here we report that PTEN plays an important role in regulating mitotic timing, which is associated with increased PTEN phosphorylation in the C-terminal tail and its localization to chromatin. Pulldown analysis revealed that Plk1 physically interacted with PTEN. Biochemical studies showed that Plk1 phosphorylates PTEN in vitro in a concentration-dependent manner and that the phosphorylation was inhibited by Bi2635, a Plk1-specific inhibitor. Deletional and mutational analyses identified that Plk1 phosphorylated Ser-380, Thr-382, and Thr-383, but not Ser-385, a cluster of residues known to affect the PTEN stability. Interestingly, a combination of molecular and genetic analyses revealed that only Ser-380 was significantly phosphorylated in vivo and that Plk1 regulated the phosphorylation, which was associated with the accumulation of PTEN on chromatin. Moreover, expression of phospho-deficient mutant, but not wild-type PTEN, caused enhanced mitotic exit. Taken together, our studies identify Plk1 as an important regulator of PTEN during the cell cycle.     

Involvement of polo-like kinase 1 (Plk1) in mitotic arrest by inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase-ribosomal S6 kinase 1 (MEK-ERK-RSK1) cascade

Cell division is controlled through cooperation of different kinases. Of these, polo-like kinase 1 (Plk1) and p90 ribosomal S6 kinase 1 (RSK1) play key roles. Plk1 acts as a G(2)/M trigger, and RSK1 promotes G(1) progression. Although previous reports show that Plk1 is suppressed by RSK1 during meiosis in Xenopus oocytes, it is still not clear whether this is the case during mitosis or whether Plk1 counteracts the effects of RSK1. Few animal models are available for the study of controlled and transient cell cycle arrest. Here we show that encysted embryos (cysts) of the primitive crustacean Artemia are ideal for such research because they undergo complete cell cycle arrest when they enter diapause (a state of obligate dormancy). We found that Plk1 suppressed the activity of RSK1 during embryonic mitosis and that Plk1 was inhibited during embryonic diapause and mitotic arrest. In addition, studies on HeLa cells using Plk1 siRNA interference and overexpression showed that phosphorylation of RSK1 increased upon interference and decreased after overexpression, suggesting that Plk1 inhibits RSK1. Taken together, these findings provide insights into the regulation of Plk1 during cell division and Artemia diapause cyst formation and the correlation between the activity of Plk1 and RSK1.     

Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP–Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.     

Spatial and temporal expression patterns of chitinase genes in developing zebrafish embryos

Chitinases and chitinase like proteins play an important role in mammalian immunity and functions in early zebrafish development have been suggested. Here we report identification of six zebrafish chitinases and chitinase like proteins (called CHIA.1–6) belonging to the glycoside hydrolase family 18, and determine their spatial and temporal expression at 10 stages of zebrafish development.CHIA.4 is highly maternally expressed and it is expressed 100 fold above any other CHIA gene at zygote through to blastula stage. Later, after the maternal to zygotic transition, CHIA.4 expression decreases to the same level as CHIA.5 and CHIA.6. Subsequently, CHIA.1, CHIA.2, CHIA.3 and CHIA.4, CHIA.5, CHIA.6 each follow distinct paths in terms of expression levels.Until 4 days post fertilization the spatial expression patterns of all six CHIA genes overlap extensively, with expression detected predominantly in vascular, ocular and intestinal tissues. At 5 days post fertilization CHIA.1, CHIA.2 and CHIA.3 are expressed almost exclusively in the stomach, whereas CHIA.4, CHIA.5 and CHIA.6 are also prominently expressed in the liver. These different expression patterns may contribute to the establishment of a basis on which functional analysis in older larvae may be founded.     

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. We show that polyhomeotic(proximal) but not polyhomeotic(distal) is necessary for mitosis. To test if other PcG genes have roles in mitosis, we examined embryos derived from heterozygous PcG mutant females for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. We propose that these mitotic defects in PcG mutants delay cell cycle progression. We discuss possible mitotic roles for PcG proteins, and suggest that delays in cell cycle progression might lead to failure of maintenance.     

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish

The pcdhα/CNR gene comprises a diverse array of neuronal cell-surface proteins of the cadherin superfamily, although very little is known about their role in neural development. Here we provide the first in-depth characterization of pcdh1α in zebrafish. Whole-mount immunocytochemistry demonstrates that a large proportion of endogenous cytoplasmic domain immunoreactivity is present in the nucleus, suggesting that endoproteolytic cleavage and nuclear translocation of the intracellular domain are important aspects of pcdh1α activity in vivo. Using whole-mount immunocytochemistry and BAC-based expression of Pcdh1α-GFP fusion proteins, we find that Pcdh1α does not appear to form stable, synaptic puncta at early stages of synaptogenesis. We also demonstrate that the presence of the Pcdh1α cytoplasmic domain is essential for normal function. Truncation of Pcdh1α proteins, using splice-blocking antisense morpholinos to prevent the addition of the common intracellular domain to the entire pcdh1α cluster, results in neuronal apoptosis throughout the developing brain and spinal cord, demonstrating an essential role for pcdh1α in early neural development. This cell death phenotype can be attenuated by the expression of a soluble Pcdh1α cytoplasmic domain.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.