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Dramatic cellular reorganization in mitosis critically depends on the timely and temporal phosphorylation of a broad range of proteins, which is mediated by the activation of the mitotic kinases and repression of counteracting phosphatases. The mitosis-to-interphase transition, which is termed mitotic exit, involves the removal of mitotic phosphorylation by protein phosphatases. Although protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) drive this reversal in animal cells, the phosphatase network associated with ordered bulk dephosphorylation in mitotic exit is not fully understood. Here, we describe a new mitotic phosphatase relay in which Wip1/PPM1D phosphatase activity is essential for chromosomal passenger complex (CPC) translocation to the anaphase central spindle after release from the chromosome via PP1-mediated dephosphorylation of histone H3T3. Depletion of endogenous Wip1 and overexpression of the phosphatase-dead mutant disturbed CPC translocation to the central spindle, leading to failure of cytokinesis. While Wip1 was degraded in early mitosis, its levels recovered in anaphase and the protein functioned as a Cdk1-counteracting phosphatase at the anaphase central spindle and midbody. Mechanistically, Wip1 dephosphorylated Thr-59 in inner centromere protein (INCENP), which, subsequently bound to MKLP2 and recruited other components to the central spindle. Furthermore, Wip1 overexpression is associated with the overall survival rate of patients with breast cancer, suggesting that Wip1 not only functions as a weak oncogene in the DNA damage network but also as a tumor suppressor in mitotic exit. Altogether, our findings reveal that sequential dephosphorylation of mitotic phosphatases provides spatiotemporal regulation of mitotic exit to prevent tumor initiation and progression.

Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy, suggesting that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

Background Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

Although human embryonic stem cells (hESCs) are equipped with highly effective machinery for the maintenance of genome integrity, the frequency of genetic aberrations during long-term in vitro hESC culture has been a serious issue that raises concerns over their safety in future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in the late-passaged hESCs (LP-hESCs) in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs). Through high-resolution genome-wide approaches and by following transcriptome analysis, we found that LP-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2 (targeting protein for Xklp2), a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stability, misaligned chromosomes, and polyploidy. This data suggests that the amplification and increased transcription of the TPX2 gene at 20q11.21 could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis., The proteins of the chromosomal passenger complex help chromosomes condense before cell division, but how this complex arrives at chromosomes was not known. Here the authors show that PRMT6 methylates histone H3 to recruit the chromosomal passenger complex.

BACKGROUND AND PURPOSE Chromosomal instability is not only a hallmark of cancer, but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed. EXPERIMENTAL APPROACH The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis, and spindle assembly checkpoint were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analyzed. KEY RESULTS Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12-treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, growth inhibition of HCC cells. In addition, expression of the Mps1-encoding TTK gene was associated with poor overall survival of HCC patients. CONCLUSION AND IMPLICATIONS TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach to liver cancer.

Bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl)propane] is one of the most prevalent synthetic environmental estrogens; as an endocrine disruptor, it is associated with endocrine-related cancers including breast, ovarian, and prostate. However, the mechanisms by which BPA contributes to carcinogenesis are unclear. This study aims to clarify its toxic effects on mitotic cells and investigate the molecular mechanism. In vitro effects of BPA on mitotic progression were examined by performing experiments on HeLa cells. Proteins involved in mitotic processes were detected by Western blot, live cell imaging, and immunofluorescence staining. The results showed that BPA increased chromosomal instability by perturbing mitotic processes such as bipolar spindle formation and spindle microtubule attachment to the kinetochore. BPA prolonged mitotic progression by disturbing spindle attachment and concomitant activating spindle assembly checkpoint (SAC). Mechanistically, BPA interfered proper localization of HURP to the proximal ends of spindle microtubules, Kif2a to the minus ends of spindle microtubules, and TPX2 on the mitotic spindle. This mislocalization of microtubule associated proteins (MAPs) is postulated to lead to spindle attachment failure. Furthermore, BPA caused multipolar spindle by inducing centriole overduplication and premature disengagement. Although BPA acts as an estrogen receptor (ER) agonist, mitotic defects caused by BPA occurred in an ER-independent manner. Our findings indicate that BPA may stimulate carcinogenesis not only by acting as an endocrine disruptor but also by increasing chromosomal instability during mitosis.

Larrea nitida Cav. (LNC), which belongs to the family Zygophyllaceae, is widely indigenous and used in South America to treat various pathological conditions. It contains the antioxidant and antiinflammatory but toxic nordihydroguaiaretic acid (NDGA) as well as O-methylated metabolite of NDGA (MNDGA) as bioactive compounds. The hepatic metabolism-based toxicological potential of extracts of LNC (LNE), NDGA, and MNDGA has not previously been reported. The present study aimed to characterize the phase I and phase II hepatic metabolism and reactive intermediates of LNE, NDGA, and MNDGA and their effects on the major drug-metabolizing enzymes in vitro and ex vivo. A methanol extract of LNC collected from Chile as well as NDGA and MNDGA isolated from LNE were subjected to metabolic stability assays in liver microsomes in the presence of the cofactors reduced nicotinamide dinucleotide phosphate (NADPH) and/or uridine 5'-diphosphoglucuronic acid (UDPGA). Cytochrome P450 (CYP) inhibition assays were performed using CYP isozyme-specific model substrates to examine the inhibitory activities of LNE, NDGA, and MNDGA, which were expressed as % inhibition and IC50 values. Ex vivo CYP induction potential was investigated in the liver microsomes prepared from the rats intraperitoneally administered with LNE. Glutathione (GSH) adduct formation was monitored by LC-MS3 analysis of the microsomal incubation samples with either NDGA or MNDGA and an excess of GSH to determine the formation of electrophilic reactive intermediates. Both NDGA and MNDGA were stable to NADPH-dependent phase I metabolism, but labile to glucuronide conjugation. LNE, NDGA, and MNDGA showed significant inhibitory effects on CYP1A2, 2C9, 2D6, and/or 3A4, with IC50 values in the micromolar range. LNE was found to be a CYP1A2 inducer in ex vivo rat experiments, and mono- and di-GSH adducts of both NDGA and MNDGA were identified by LC-MS3 analysis. Our study suggests that hepatic clearance is the major elimination route for the lignans NDGA and MNDGA present in LNE. These lignans may possess the ability to modify biomacromolecules via producing reactive intermediates. In addition, LNE, NDGA, and MNDGA are found to be inhibitors for various CYP isozymes such as CYP2C9 and 3A4. Thus, the consumption of LNC as an herbal preparation or NDGA may cause metabolism-driven herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd.

The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17β-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John Wiley & Sons, Ltd.

Spindle microtubules (MTs) capture kinetochores (KTs) on the centromere sequence of sister chromatids to align at the mitotic equator and segregate toward spindle poles during mitosis. For efficient chromosome capture, KTs initially attach to the lateral surface of a MT, providing a considerably larger contact surface than the MT tip. A sequential change of KT composition upon spindle attachment enables a conversion from lateral to stable end-on attachment. However, the molecular link between spindle dynamics and KT composition is not fully understood. Here, we report that Ska1 and DDA3 act as molecular linkers in the interplay between KTs and spindle dynamics. After recruitment of Kif2a onto the mitotic spindle by DDA3, Ska1 targets Kif2a to the minus-end of spindle MTs and facilitates spindle dynamics. Furthermore, DDA3 targets Ska1 to KTs to stabilize end-on attachment. Thus, our findings identified a definite regulatory mechanism of the search and capture process for stable spindle attachment through cross-talk between spindle dynamics and KT composition mediated by DDA3 and Ska1.

Phytoestrogens possess beneficial effects in the management of menopausal symptoms with few side effects. Soybeans are major natural sources of isoflavones, with high estrogen receptor (ER)-β selectivity. The objective of this study therefore was to develop a solvent-mediated extraction method for soybean germinated embryos (SGEs) and to investigate the biological activities of the extract. Ethanolic extraction yielded the SGE extract (SGEE), which had a unique composition of biologically active aglycones and soyasaponins. SGEE showed a proliferative effect in MCF7 cells and ERβ-selective transcriptional activities in human embryonic kidney cells. In addition, oral administration of SGEE to ovariectomized rats resulted in the induction of ERβ and estrogen-responsive genes in the uterus and a decrease in tail skin temperature and uterus weight. Our data suggest that germination and ethanolic extraction are effective measures for producing isoflavone-rich food supplements, which may be useful as alternative menopausal hormone therapy.

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug&ndash, herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.

Estrogen receptors are activated by the hormone estrogen and they control cell growth by altering gene expression as a transcription factor. So far two estrogen receptors have been found: ERα and ERβ. Estrogen receptors are also implicated in the development and progression of breast cancer. Here, we found that ERα localized on the spindle and spindle poles at the metaphase during mitosis. Depletion of ERα generated unaligned chromosomes in metaphase cells and lagging chromosomes in anaphase cells in a transcription-independent manner. Furthermore, the levels of β-tubulin and γ-tubulin were reduced in ERα-depleted cells. Consistent with this, polymerization of microtubules in ERα-depleted cells and turnover rate of α/β-tubulin were decreased than in control cells. We suggest that ERα regulates chromosome alignment and spindle dynamics by stabilizing microtubules during mitosis.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent risk factor among tobacco-related carcinogens in lung cancer progression and outcomes. Although genetic mutations and chromosome instability have been detected in NNK-induced lung tumors, the oncogenic mechanisms of NNK are not fully understood. Here, we show that NNK increases chromosomal instability by disrupting spindle microtubule (MT) attachment to the kinetochore (KT) and spindle dynamics. Mechanistically, NNK blocks the targeting of p53 to the centrosome during mitosis, leading to chromosome alignment defects in metaphase. Therefore, lung cancer cells with wild-type p53, such as A594 and H226B, are more resistant to the NNK treatment than p53-mutant lung cancer cells, such as A1299 and H226Br. Although NNK does not affect the levels or transcriptional activity of p53, the reduction of the p53 level at the centrosome exacerbates the NNK-induced chromosome alignment defect in A549 and H226B cells. Therefore, p53 protects against NNK-induced chromosome instability by modulating the function of centrosome-localized p53 and not by modulating transcriptional activity. We conclude that NNK may increase the risk of lung cancer progression and poorer outcomes in patients with p53 mutations by perturbing proper mitotic progression and chromosome integrity.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is overexpressed in most human cancers and has been described as being involved in the progression of several human malignancies via the inhibition of protein phosphatase 2A (PP2A) activity toward c-Myc. However, with the exception of this role, the cellular function of CIP2A remains poorly understood. On the basis of yeast two-hybrid and coimmunoprecipitation assays, we demonstrate here that NIMA (never in mitosis gene A)-related kinase 2 (NEK2) is a binding partner for CIP2A. CIP2A exhibited dynamic changes in distribution, including the cytoplasm and centrosome, depending on the cell cycle stage. When CIP2A was depleted, centrosome separation and the mitotic spindle dynamics were impaired, resulting in the activation of spindle assembly checkpoint signaling and, ultimately, extension of the cell division time. Our data imply that CIP2A strongly interacts with NEK2 during G2/M phase, thereby enhancing NEK2 kinase activity to facilitate centrosome separation in a PP1- and PP2A-independent manner. In conclusion, CIP2A is involved in cell cycle progression through centrosome separation and mitotic spindle dynamics.

Carbon nanofilaments (CNFs) could be synthesized using C₂H₂/H₂ as source gases and SF6 as an incorporated additive gas under thermal chemical vapor deposition system. Ni powders were used as the catalyst for the formation of the CNFs. During the initial deposition stage, the initiation of the CNFs on the Ni catalyst was investigated. The geometries of the as-grown CNFs on Ni catalyst were strongly dependent on the size and/or the shape of Ni catalyst. Small size catalyst (

Chromosomal instability is not only a hallmark of cancer but also an attractive therapeutic target. A diverse set of mitotic kinases maintains chromosomal stability. One of these is monopolar spindle 1 (Mps1, also known as TTK), which is essential for chromosome alignment and for the spindle assembly checkpoint (SAC). Pharmacological inhibition of Mps1 has been suggested as a cancer therapeutic; however, despite the existence of a novel Mps1 inhibitor, TC Mps1 12, no such studies have been performed.The effects of TC Mps1 12 on cell viability, chromosome alignment, centrosome number, mitotic duration, apoptosis and SAC were determined in hepatocellular carcinoma (HCC) cells. In addition, the association of Mps1 expression with the overall survival of HCC patients was analysed.Treatment of human HCC cells with TC Mps1 12 led to chromosome misalignment and missegregation, and disorganization of centrosomes. Even in the presence of these errors, TC Mps1 12-treated cells overrode the SAC, resulting in a shortened mitotic duration and mitotic slippage. This mitotic catastrophe triggered apoptosis and, finally, inhibited the growth of HCC cells. In addition, the expression of the Mps1-encoding TTK gene was associated with poor overall survival of HCC patients.TC Mps1 12 results in the accumulation of chromosomal instabilities and mitotic catastrophe in HCC cells. Overall, these data demonstrate that the inhibition of Mps1 kinase using TC Mps1 12 is a promising therapeutic approach for liver cancer.

Larrea nitida Cav. (LNC), which belongs to the family Zygophyllaceae, is widely indigenous and used in South America to treat various pathological conditions. It contains the antioxidant and antiinflammatory but toxic nordihydroguaiaretic acid (NDGA) as well as O-methylated metabolite of NDGA (MNDGA) as bioactive compounds. The hepatic metabolism-based toxicological potential of extracts of LNC (LNE), NDGA, and MNDGA has not previously been reported. The present study aimed to characterize the phase I and phase II hepatic metabolism and reactive intermediates of LNE, NDGA, and MNDGA and their effects on the major drug-metabolizing enzymes in vitro and ex vivo. A methanol extract of LNC collected from Chile as well as NDGA and MNDGA isolated from LNE were subjected to metabolic stability assays in liver microsomes in the presence of the cofactors reduced nicotinamide dinucleotide phosphate (NADPH) and/or uridine 5'-diphosphoglucuronic acid (UDPGA). Cytochrome P450 (CYP) inhibition assays were performed using CYP isozyme-specific model substrates to examine the inhibitory activities of LNE, NDGA, and MNDGA, which were expressed as % inhibition and IC

Na-Kyung Han,1,* Dae Hwan Shin,1,* Jung Seok Kim,1 Kwon Yeon Weon,2 Chang-Young Jang,1 Jin-Seok Kim1 1Research Center for Cell Fate Control (RCCFC) and College of Pharmacy, Sookmyung Women’s University, Seoul, 2College of Pharmacy, Catholic University of Daegu, Gyeongbuk, Korea *These authors contributed equally tothis work Abstract: Investigation of potential therapeutics for targeting breast cancer stem cells (BCSCs) is important because these cells are regarded as culprit of breast cancer relapse. Accomplishing this kind of strategy requires a specific drug-delivery system using the distinct features of liposomes. Studies on targeted liposomal delivery systems have indicated the conjugation of hyaluronan (HA), a primary ligand for CD44 surface markers, as an appropriate method for targeting BCSCs. For this study, enriched BCSCs were obtained by culturing MCF-7 breast cancer cells in nonadherent conditions. The enriched BCSCs were challenged with HA-conjugated liposomes encapsulating gemcitabine (2, 2-difluoro-2-deoxycytidine, GEM). In vitro study showed that the HA-conjugated liposomes significantly enhanced the cytotoxicity, anti-migration, and anti-colony formation abilities of GEM through targeting of CD44 expressed on BCSCs. In pharmacokinetic study, area under the drug concentration vs time curve (AUC) of the immunoliposomal GEM was 3.5 times higher than that of free GEM, indicating that the HA-conjugated liposomes enhanced the stability of GEM in the bloodstream and therefore prolonged its half-life time. The antitumor effect of the immunoliposomal GEM was 3.3 times higher than that of free GEM in a xenograft mouse model, probably reflecting the unique targeting of the CD44 receptor by HA and the increased cytotoxicity and stability through the liposomal formulation. Furthermore, marginal change in body weight demonstrated that the use of liposomes considerably reduced the systemic toxicity of GEM on normal healthy cells. Taken together, this study demonstrates that HA-conjugated liposomes encapsulating GEM show promise for the therapy of breast cancer in vitro and in a xenograft model by targeting the BCSCs. Keywords: breast cancer stem cells, targeting, CD44 surface marker, EPR effect, drug delivery system

Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

It has been reported that ribosomal protein S3 (rpS3) functions as a ribosomal protein, a DNA repair endonuclease, a proapoptotic protein, and an essential subunit of the native NF-κB complex. However, it is unknown how rpS3 induces apoptosis in response to extracellular stresses. We report here that rpS3 sensitizes genotoxic stress-induced apoptosis by activating JNK through a caspase dependent manner. This apoptotic effect was shown to result from the physical interaction between rpS3 and TRADD, as assessed by coimmunoprecipitation. Moreover, GFP-rpS3 colocalized with TRADD around the plasma membrane and in the cytoplasm during apoptosis. Thus, rpS3 appears to be recruited to the death-inducing signaling complex (DISC) to induce apoptosis by interacting TRADD in response to extracellular stresses. Based on the findings of this study, we concluded that rpS3 is recruited to the DISC and plays a critical role in both genotoxic stress and cytokine induced apoptosis.

Mitotic kinases orchestrate cell cycle processes by phosphorylation of cell cycle regulators. DDA3, a spindle-associated phosphor-protein, is a substrate of mitotic kinases that control chromosome movement and spindle microtubule (MT) dynamics. Through a mass spectrometry analysis, we identified phosphorylation sites on the endogenous mitotic DDA3, which include Ser22, Ser65, Ser70, and Ser223. Phosphorylation of these residues converts interphase form of DDA3 to mitotic form by changing its biochemical activity, as unphosphorylated DDA3 processed both the MT polymerizing and bundling activities, whereas phosphor-mimic mutants lost both activities, only retaining the MT-binding activity. We found that mitotic kinases, such as Cdk1, Aurora A, and Plk1, phosphorylate DDA3 in vitro. Whereas Cdk1 and Aurora A negatively regulate MT-polymerizing and MT-bundling activities, Plk1 does not affect these activities. Interestingly, the phosphorylation of DDA3 by Aurora A and Plk1 inhibits the phosphorylation by other kinases, indicating that sequential phosphorylation is important for the regulation of DDA3 function. We conclude that kinases control the function of DDA3 in the cell cycle by regulating its MT-polymerizing/bundling activities through sequential phosphorylation.

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue.

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

Through a convergence of functional genomic and proteomic studies, we identify Bora as a previously unknown cell cycle protein that interacts with the Plk1 kinase and the SCF–β-TrCP ubiquitin ligase. We show that the Bora protein peaks in G2 and is degraded by proteasomes in mitosis. Proteolysis of Bora requires the Plk1 kinase activity and is mediated by SCF–β-TrCP. Plk1 phosphorylates a conserved DSGxxT degron in Bora and promotes its interaction with β-TrCP. Mutations in this degron stabilize Bora. Expression of a nondegradable Bora variant prolongs the metaphase and delays anaphase onset, indicating a physiological requirement of Bora degradation. Interestingly, the activity of Bora is also required for normal mitotic progression, as knockdown of Bora activates the spindle checkpoint and delays sister chromatid segregation. Mechanistically, Bora regulates spindle stability and microtubule polymerization and promotes tension across sister kinetochores during mitosis. We conclude that tight regulation of the Bora protein by its synthesis and degradation is critical for cell cycle progression.

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.

Spindle dynamics drives chromosome movement and mitotic progression during mitosis. Microtubule (MT)-associated proteins (MAPs) regulate MT stabilization/destabilization and MT polymerization/depolymerization for congression of sister chromatids at the mitotic equator and subsequent segregation toward the spindle poles. Here, we identified ANKRD53 as a novel DDA3-interacting protein through proteomic analysis. Based on expression profiles, ANKRD53 is phosphorylated by mitotic kinases during mitosis. In ANKRD53-depleted HeLa cells, the progression of mitosis was delayed and the number of unaligned chromosomes increased substantially. In addition, spindle MT polymerization decreased and the spindle assembly checkpoint (SAC) was concomitantly activated by the decreased spindle dynamics in ANKRD53-depleted cells. Although ANKRD53 is recruited to the mitotic spindle by DDA3, it counteracts the activity of DDA3 for spindle MT polymerization. Furthermore, ANKRD53 depletion increased the number of bi-nuclei and polylobed nuclei. Thus, ANKRD53 is recruited to the mitotic spindle by DDA3 and acts as a regulator of spindle dynamics and cytokinesis.

Phytoestrogens are selective estrogen receptor modulators (SERMs) with potential for use in hormone replacement therapy (HRT) to relieve peri/postmenopausal symptoms. This study was aimed at elucidating the molecular mechanisms underlying the SERM properties of the extract of Korean-grown Opuntia ficus-indica (KOFI). The KOFI extract induced estrogen response element (ERE)-driven transcription in breast and endometrial cancer cell lines and the expression of endogenous estrogen-responsive genes in breast cancer cells. The flavonoid content of different KOFI preparations affected ERE-luciferase activities, implying that the flavonoid composition likely mediated the estrogenic activities in cells. Oral administration of KOFI decreased the weight gain and levels of both serum glucose and triglyceride in ovariectomized (OVX) rats. Finally, KOFI had an inhibitory effect on the 17β-estradiol-induced proliferation of the endometrial epithelium in OVX rats. Our data demonstrate that KOFI exhibited SERM activity with no uterotrophic side effects. Therefore, KOFI alone or in combination with other botanical supplements, vitamins, or minerals may be an effective and safe alternative active ingredient to HRTs, for the management of postmenopausal symptoms. Copyright © 2016 John WileySons, Ltd.

Aurora B activation is triggered at the mitotic entry and required for proper microtubule-kinetochore attachment at mitotic phase. Therefore, Aurora B should be in inactive form in interphase to prevent aberrant cell cycle progression. However, it is unclear how the inactivation of Aurora B is sustained during interphase. In this study, we find that IK depletion-induced mitotic arrest leads to G2 arrest by Aurora B inhibition, indicating that IK depletion enhances Aurora B activation before mitotic entry. IK binds to Aurora B, and colocalizes on the nuclear foci during interphase. Our data further show that IK inhibits Aurora B activation through recruiting PP2A into IK and Aurora B complex. It is thus believed that IK, as a scaffold protein, guides PP2A into Aurora B to suppress its activity in interphase until mitotic entry.

Active turnover of spindle microtubules (MTs) for the formation of a bi-orientated spindle, chromosome congression and proper chromosome segregation is regulated by MT depolymerases such as the kinesin-13 family and the plus-end-tracking proteins (+TIPs). However, the control mechanisms underlying the spindle MT dynamics that are responsible for poleward flux at the minus end of MTs are poorly understood. Here, we show that Mdp3 (also known as MAP7D3) forms a complex with DDA3 (also known as PSRC1) and controls spindle dynamics at the minus end of MTs by inhibiting DDA3-mediated Kif2a recruitment to the spindle. Aberrant Kif2a activity at the minus end of spindle MTs in Mdp3-depleted cells decreased spindle stability and resulted in unaligned chromosomes in metaphase, lagging chromosomes in anaphase, and chromosome bridges in telophase and cytokinesis. Although they play opposing roles in minus-end MT dynamics, acting as an MT destabilizer and an MT stabilizer, respectively, DDA3 and Mdp3 did not affect the localization of each other. Thus, the DDA3 complex orchestrates MT dynamics at the MT minus end by fine-tuning the recruitment of Kif2a to regulate minus-end MT dynamics and poleward MT flux at the mitotic spindle.

The centrosome is an important cellular organelle which nucleates microtubules (MTs) to form the cytoskeleton during interphase and the mitotic spindle during mitosis. The Cep290 is one of the centrosomal proteins and functions in cilia formation. Even-though it is in the centrosome, the function of Cep290 in mitosis had not yet been evaluated. In this study, we report a novel function of Cep290 that is involved in spindle positioning. Cep290 was identified as an interacting partner of DDA3, and we confirmed that Cep290 specifically localizes in the mitotic centrosome. Depletion of Cep290 caused a reduction of the astral spindle, leading to misorientation of the mitotic spindle. MT polymerization also decreased in Cep290-depleted cells, suggesting that Cep290 is involved in spindle nucleation. Furthermore, DDA3 stabilizes and transports Cep290 to the centrosome. Therefore, we concluded that DDA3 controls astral spindle formation and spindle positioning by targeting Cep290 to the centrosome.

It is known that mammalian rpS3 functions as a DNA repair endonuclease and ribosomal protein S3. It was also observed that several ribosomal proteins or DNA repair enzymes are related to apoptosis. We report here a third function of rpS3, induction of apoptosis. The localization of green fluorescent protein (GFP)-rpS3 is changed to the nuclear membrane when lymphocytic cells undergo rpS3-induced apoptosis. Transient expression of GFP-rpS3 activates caspase-8/caspase-3 and sensitizes cytokine-induced apoptosis. Deletion analysis reveals that the two functions of rpS3, DNA repair and apoptosis, use independent functional domains.

When a ribosome complex is stalled during the translation elongation process in eukaryotes, the mono-ubiquitination of Rps3 has recently been shown to be critical to ribosome quality control. We have discovered that the regulatory role of Rps3 mono-ubiquitination is controlled by a deubiquitinase. We also showed that an autophagic signal appears to be coupled to the mono-ubiquitination of Rps3p through the entrance of Ubp3p into the autophagosome in yeasts. The mono-ubiquitination of the Rps3 protein is tightly modulated by reciprocal action between the Hel2p E3 ligase and the Ubp3p deubiquitinase in yeasts and the reciprocal action between the RNF123 E3 ligase and the USP10 deubiquitinase in mammalian cells. We also found that the Ubp3p/USP10 deubiquitinases critically modulate Hel2p/RNF123-mediated Rps3p mono-ubiquitination. In addition, we found that Hel2p/RNF123 and Ubp3p/USP10 appeared to be differently localized in the ribosome complex after ultraviolet irradiation. Together, our results support a model in which coordinated ubiquitination and deubiquitination activities can finely balance the level of regulatory Rps3p mono-ubiquitination in ribosome-associated quality control and autophagy processes.

Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.

Sirt3, one of mammalian sirtuins is a prominent mitochondrial deacetylase that controls mitochondrial oxidative pathways and the rate of reactive oxygen species. Sirt3 also regulates energy metabolism by deacetylating enzymes involved in the metabolic pathway related with lifespan. We report here a novel function of Sirt3 which was found to be involved in mitosis. Depletion of the Sirt3 protein generated unaligned chromosomes in metaphase which caused mitotic arrest by activating spindle assembly checkpoint (SAC). Furthermore, the shape and the amount of the spindles in Sirt3 depleted cells were abnormal. Microtubule (MT) polymerization also increased in Sirt3 depleted cells, suggesting that Sirt3 is involved in spindle dynamics. However, the level of acetylated tubulin was not increased significantly in Sirt3 depleted cells. The findings collectively suggest that Sirt3 is not a tubulin deacetylase but regulates the attachment of spindle MTs to the kinetochore and the subsequent chromosome alignment by increasing spindle dynamics.

KH2PO4 single crystals have been studied by employing complex impedance measurements in view of the domain freezing effect. As a result, distinct behaviors of the anisotropic domain-wall dynamics in the activation energy of domain freezing and the Vogel–Fulcher temperature before and after proton irradiation have been identified in the anisotropic crystal structure.

Abnormal activation of the Wnt/β-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on β-catenin dependent Wnt pathway. TSL suppressed β-catenin/T-cell factor transcriptional activity and down-regulated β-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3β. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of β-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the β-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/β-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.

The dissipative and dispersive properties of Hermite methods are analyzed by a modified equation approach and by direct computation of the dispersion relations for the discrete modes of the scheme. The two approaches lead to the same results for well-resolved modes but are quantitatively different at the finest scales. The resolution requirements, obtained from the analysis, for Hermite schemes are compared to those of typical high-resolution difference formulas. The results from the analysis are also used to predict the resolution requirements for a simulation at Re � 3600. The validity of the prediction is confirmed by numerical experiments.

5-Lipoxygenase (5-LO) catalyzes the formation of two major groups of leukotrienes, leukotriene B4 and cysteinyl leukotrienes (CysLTs), and it has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in osteoclastogenesis has not been investigated. In this study, we used mouse bone marrow–derived macrophages (BMMs) to show that 5-LO inhibitor suppresses RANKL-induced osteoclast formation. Inhibition of 5-LO was associated with impaired activation of multiple signaling events downstream of RANK, including ERK and p38 phosphorylation, and IκB degradation, followed by a decrease in NFATc1 expression. Ectopic overexpression of a constitutively active form of NFATc1 partly rescued the antiosteoclastogenic effect of 5-LO inhibitor. The knockdown of 5-LO in BMMs also resulted in a significant reduction in RANKL-induced osteoclast formation, accompanied by decreased expression of NFATc1. Similar effects were shown with CysLT receptor (CysLTR)1/2 antagonist and small RNA for CysLTR1 in BMMs, indicating the involvement of CysLT and CysLTR1 in 5-LO–mediated osteoclastogenesis. Finally, 5-LO inhibitor suppressed LPS-induced osteoclast formation and bone loss in the in vivo mouse experiments, suggesting a potential therapeutic strategy for treating diseases involving bone destruction. Taken together, the results of this study demonstrate that 5-LO is a key mediator of RANKL-induced osteoclast formation and possibly a novel therapeutic target for bone-resorption diseases.

The human ribosomal protein S3 (rpS3) has multi-functions such as translation, DNA repair and apoptosis. These multiple functions are regulated by post-translational modifications including phosphorylation, methylation and sumoylation. We report here a novel function of rpS3 that is involved in mitosis. When we examined localization of ribosomal proteins in mitosis, we found that rpS3 specifically localizes on the mitotic spindle. Depletion of the rpS3 proteins caused mitotic arrest during the metaphase. Furthermore, the shape of the spindle and chromosome movement in the rpS3 depleted cell was abnormal. Microtubule (MT) polymerization also decreased in rpS3 depleted cells, suggesting that rpS3 is involved in spindle dynamics. Therefore, we concluded that rpS3 acts as a microtubule associated protein (MAP) and regulates spindle dynamics during mitosis.