Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

<p>Abstract</p> <p>Background</p> <p>Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of <it>Curcuma longa</it>. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy.</p> <p>Methods</p> <p>Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an <it>in vitro </it>APC activity assay.</p> <p>Results</p> <p>We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells.</p> <p>Conclusions</p> <p>We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin.</p

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis

APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Faithful chromosome segregation during mitosis depends on the Spindle Assembly Checkpoint (SAC) that monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the Anaphase Promoting Complex/Cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important to understand how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator Cdc20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated Cdc20 remain ‘locked’ onto the APC/C, which prevents the ubiquitylation and degradation of Cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of Cdc20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores

GPS-ARM: Computational Analysis of the APC/C Recognition Motif by Predicting D-Boxes and KEN-Boxes

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org

A quantitative systems view of the spindle assembly checkpoint

The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway

Natural multi-occurrence of mycotoxins in rice from Niger State, Nigeria

Twenty-one rice samples from field (ten), store (six) and market (five) from the traditional rice-growing areas of Niger State, Nigeria were analysed for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B1 (FB1) and B2 (FB2), and patulin (PAT) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) respectively. T-2 toxin was determined using TLC only. AFs were detected in all samples, at total AF concentrations of 28–372 μg/kg. OTA was found in 66.7% of the samples, also at high concentrations (134–341 μg/kg) that have to be considered as critical levels in aspects of nephrotoxicity. ZEA (53.4%), DON (23.8), FB1 (14.3%) and FB2 (4.8%) were also found in rice, although at relatively low levels. T-2 toxin was qualitatively detected by TLC in only one sample. Co-contamination with AFs, OTA, and ZEA was very common, and up to five mycotoxins were detected in a single sample. The high AF and OTA levels as found in rice in this study are regarded as unsafe, and multi-occurrences of mycotoxins in the rice samples with possible additive or synergistic toxic effects in consumers raise concern with respect to public health

Spindle Assembly Checkpoint Protein Dynamics Reveal Conserved and Unsuspected Roles in Plant Cell Division

Background: In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle. Methodology/Principal Findings: We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of 'wait anaphase', plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants. Conclusions/Significance: We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division

T-2 toksin - pojavnost i toksičnost u peradi

T-2 toxin is the most toxic type A trichothecene mycotoxin. It is the secondary metabolite of the Fusarium fungi, and is common in grain and animal feed. Toxic effects have been shown both in experimental animals and in livestock. It has been implicated in several outbreaks of human mycotoxicoses. Toxic effects in poultry include inhibition of protein, DNA, and RNA synthesis, cytotoxicity, immunomodulation, cell lesions in the digestive tract, organs and skin, neural disturbances and low performance in poultry production (decreased weight gain, egg production, and hatchability). Concentrations of T-2 toxin in feed are usually low, and its immunosuppressive effects and secondary infections often make diagnosis difficult. If at the onset of the disease, a change in diet leads to health and performance improvements in animals, this may point to mycotoxin poisoning. Regular control of grain and feed samples is a valuable preventive measure, and it is accurate only if representative samples are tested. This article reviews the incidence and toxic effects of T-2 toxin in poultry.T-2 toksin je najtoksičniji predstavnik trikotecenskih mikotoksina tipa A. On je sekundarni produkt metabolizma plijesni roda Fusarium i često je prisutan u žitaricama i hrani za životinje. Štetni učinci uočeni su u eksperimentalnih životinja i životinja u uzgoju. On se povezuje s pojavom bolesti ljudi od mikotoksikoza. Učinci toksina u peradi su višestruki: inhibicija sinteze proteina, DNA i RNA, citotoksični učinak, imunomodulatorni učinak, oštećenje stanica probavnog sustava, organa i kože, živčani poremećaji te pad proizvodnih karakteristika u uzgoju peradi (slabiji prirast, pad nesivosti i valivosti). Koncentracije T-2 toksina u hrani redovito su vrlo malene, a zbog imunosupresivnog djelovanja toksina te istodobne sekundarne infekcije bolest se često teško dijagnosticira. Pri pojavi bolesti promjenom hrane može doći do poboljšanja zdravstvenog stanja, što tako|er upućuje na moguće trovanje mikotoksinima. Redovita kontrola uzoraka žitarica i hrane za životinje jedna je od preventivnih mjera, a detekcija mikotoksina u žitaricama i hrani pouzdana je samo ako se ispituje reprezentativan uzorak. U radu su opisani učestalost i toksični učinci T-2 toksina u peradi

Vulnerability of Polarised Intestinal Porcine Epithelial Cells to Mycotoxin Deoxynivalenol Depends on the Route of Application

BACKGROUND AND AIMS: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. METHODS: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. RESULTS: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. CONCLUSIONS: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity