Kinetochore-microtubule interactions "in check" by Bub1, Bub3 and BubR1: the dual task of attaching and signalling

The spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes accomplish proper bipolar attachments to the mitotic spindle and come under tension, thereby ensuring the fidelity of chromosome segregation. Despite significant advances in our understanding of SAC signalling, a clear link between checkpoint signalling and the molecular mechanisms underlying chromosome attachment to microtubules has not been established so far. However, independent studies from many groups have interestingly found that the bone-a-fide Bub1, BubR1 and Bub3 SAC proteins are themselves required for proper kinetochoremicrotubule (K-MT) interactions. Here, we review these findings and discuss the specific contribution of each of these proteins in the regulation of K-MT attachment, taking into consideration their interdependencies for kinetochore localization as well as their relationship with other proteins with a known role in chromosome attachment and congression.This work was supported by a grant from FCT—Fundação para a Ciência e Tecnologia (POCTI/BCI/42341/2001) and by CESPU, crl—Cooperativa de Ensino Superior Politécnico e Universitário

Mutational analysis of MSX1 and PAX9 genes in Portuguese families with maxillary lateral incisor agenesis

The observation that certain patterns of tooth agenesis occur more frequently in individuals of the same family may suggest the existence of predisposing genetic factors. The aim of this study was to search for mutations in the PAX9 and MSX1 genes and to investigate their potential association with the maxillary lateral incisor agenesis (MLIA) phenotype in 12 Portuguese families, a total of 52 individuals, 12 probands and 40 relatives (eight of which had MLIA). Twenty-three of the subjects were male and 29 female with an age range of 10-75 years. The control group comprised random DNA samples of 91 Portuguese individuals. Nucleotide alterations were not detected in the coding regions of the MSX1 gene, analysed by single-strand conformation polymorphism and sequencing; in the PAX9 gene, a polymorphism was found that led to transition of G718 to C, implying a change of alanine 240 for proline. However, the differences in the frequencies of the PAX9 gene polymorphism between the probands (67 per cent) and the control population (56 per cent carrying the c allele) were not statistically significant as determined by chi-square test, and the polymorphism did not clearly segregate with the trait in the families. Aggregating the available data, there does not seem to exist a clear association between the alanine 240 for proline variant in the PAX9 gene and the MLIA phenotype. Further studies are required to clarify the basic genetics of MLIA.The authors would like to thank the families who participated in this study. We are grateful to Pedro Seada for technical support. TP was the recipient of a scholarship from Instituto Superior de Ciencias da Saude Norte/CESPU for her PhD and AS-F is the recipient of a scholarship from FCT (SFRH/BD/15910/2005)

Long-term results of a post-partum bilateral Garden 4 femoral neck fracture in a young woman with transient osteoporosis treated with closed reduction and internal fixation

Bilateral femoral neck fractures are very rare conditions. In our knowledge, only five cases were reported of bilateral acute femoral neck fracture secondary to transient osteoporosis. The present case is the first with a long-term follow up after treatment with closed reduction and internal fixation. A 32-year-old women was admitted to the emergency orthopedic clinic due to severe pain in both hips and an inability to walk one-day post-partum. Clinical examination revealed bilateral externally rotated legs and X-ray showed bilateral Garden 4 displaced subcapital fracture of the neck of the femur. The patient was operated on urgently by closed reduction and internal fixation.  Osteoporosis was found in the measurements of bone mineral density and bisphosphonate with vitamin D medication was started. 6 years after surgery the patient can carry out her daily activities without limitation. His Harris hip score was 97 points/right and 95 points/left; and the X-ray was normal regarding osteoarthritis and avascular necrosis. Internal fixation should be the primary choice for the treatment of a bilateral femoral neck fractures with a high degree of displacement in young adults even with reversible poor bone quality condition such transient osteoporosis.

Prenylated chalcone 2 ccts as an antimitotic agent and enhances the chemosensitivity of tumor cells to paclitaxel

We previously reported that prenylated chalcone 2 (PC2), the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1), induced cytotoxicity of tumor cells via disruption of p53-MDM2 interaction. However, the cellular changes through which PC2 exerts its cytotoxic activity and its antitumor potential, remain to be addressed. In the present work, we aimed to (i) characterize the effect of PC2 on mitotic progression and the underlying mechanism; and to (ii) explore this information to evaluate its ability to sensitize tumor cells to paclitaxel in a combination regimen. PC2 was able to arrest breast adenocarcinoma MCF-7 and non-small cell lung cancer NCI-H460 cells in mitosis. All mitosis-arrested cells showed collapsed mitotic spindles with randomly distributed chromosomes, and activated spindle assembly checkpoint. Live-cell imaging revealed that the compound induced a prolonged delay (up to 14 h) in mitosis, culminating in massive cell death by blebbing. Importantly, PC2 in combination with paclitaxel enhanced the effect on cell growth inhibition as determined by cell viability and proliferation assays. Our findings demonstrate that the cytotoxicity induced by PC2 is mediated through antimitotic activity as a result of mitotic spindle damage. The enhancement effects of PC2 on chemosensitivity of cancer cells to paclitaxel encourage further validation of the clinical potential of this combination

Co-silencing of human Bub3 and dynein highlights an antagonistic relationship in regulating kinetochore-microtubule attachments

We previously reported that the spindle assembly checkpoint protein Bub3 is involved in regulating kinetochore-microtubule (KT-MT) attachments. Also, Bub3 was reported to interact with the microtubule motor protein dynein. Here we examined how this interaction contributes to KT-MT attachments. Depletion of Bub3 or dynein induced misaligned chromosomes, consistent with their role in KT-MT attachments. Unexpectedly, co-silencing of both proteins partially suppressed the misalignment phenotype and restored chromosome congression. Consistent with these observations, KT-MT attachments in co-depleted cells were stable, able to drive chromosome congression, and produce inter-and intra-kinetochore stretch, indicating they are functional. We suggest that a mutual antagonism exists between Bub3 and dynein to ensure optimal KT-MT attachments. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.CESPU [02-GCQF-CICS-2011N]; FCT - Fundacao para a Ciencia e a Tecnologia [CEQUIMED-PEst-OE/SAU/UI4040/2014]; FCT [SFRH/BD/90744/2012]info:eu-repo/semantics/publishedVersio

An overview of the spindle assembly checkpoint status in oral cancer

Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20.CESPU [02-GCQF-CICS-2011N, 01-GCD-CICS-09; 02-GCD-CICS-09, 05-GCD-CICS-2011]; Fundacao para a Ciencia e a Tecnologia (FCT) [CEQUIMED-PEst-OE/SAU/UI4040/2011]

A Pyranoxanthone as a potent antimitotic and sensitizer of cancer cells to low doses of Paclitaxel

Microtubule-targeting agents (MTAs) remain a gold standard for the treatment of several cancer types. By interfering with microtubules dynamic, MTAs induce a mitotic arrest followed by cell death. This antimitotic activity of MTAs is dependent on the spindle assembly checkpoint (SAC), which monitors the integrity of the mitotic spindle and proper chromosome attachments to microtubules in order to ensure accurate chromosome segregation and timely anaphase onset. However, the cytotoxic activity of MTAs is restrained by drug resistance and/or toxicities, and had motivated the search for new compounds and/or alternative therapeutic strategies. Here, we describe the synthesis and mechanism of action of the xanthone derivative pyranoxanthone 2 that exhibits a potent anti-growth activity against cancer cells. We found that cancer cells treated with the pyranoxanthone 2 exhibited persistent defects in chromosome congression during mitosis that were not corrected over time, which induced a prolonged SAC-dependent mitotic arrest followed by massive apoptosis. Importantly, pyranoxanthone 2 was able to potentiate apoptosis of cancer cells treated with nanomolar concentrations of paclitaxel. Our data identified the potential of the pyranoxanthone 2 as a new potent antimitotic with promising antitumor potential, either alone or in combination regimens.Portugal 2020: PTDC/SAU-PUB/28736/2017 (POCI-01-0145-FEDER-028736; FCT: SFRH/BD/116167/2016/ SFRH/BD/140844/2018/ PD/00016/2012info:eu-repo/semantics/publishedVersio

Yicathins B and C and analogues: total synthesis, lipophilicity and biological activities

Natural products had always be an important source of new hits and leads in drug discovery. The marine environment has been regarded as a significant souce of novel and exquisite bioactive compounds. Yicathins B and C are two marine derived xanthones that have shown antibacterial and antifungal activities. Herein, the total synthesis of these yicathins is reported for the first time as well as six novel analogues. As marine natural products tend to bear very lipophilic scaffolds, the lipophilicity of yicathins and its analogues was evaluated using the classical octanol:water system and a biomimetic model based system. As the xanthonic nucleus is a â privileged structureâ , other biological activities were evaluated, namely antitumor and anti-inflammatory activities. An interesting anti-inflammatory activity was identified for yicathins analogues that paves the way for the design of dual activity (anti-infective and anti-inflammatory) marine inspired xanthones derivatives.This work was supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE - Programa Operacional Factores de Competitividade (POFC) programme, under the projects PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599 - Promover a ProducAo Cientifica e Desenvolvimento Tecnologico e a ConstituicAo de Redes Tematicas (3599-PPCDT)) and PTDC/SAU-PUB/28736/2017 (reference POCI-01-0145-FEDER- 028736) in the framework of the programme PT2020. D. R. P. L. is grateful for research grant PTDC/MAR-BIO/4694/2014-BI-2017-003. J. X. S. thanks the FCT PhD Programmes and Programa Operacional Capital Humano (POCH), specifically the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), reference PD/00016/2012; through the FCT and POCH for PhD grants (SFRH/BD/98105/2013 and SFRH/BD/116167/2016). The authors would like to thank Sara Cravo and Gisela Adriano for the technical support, the Centro de Apoio Cientifico e Tecnoloxico a Investigation (C.A.C.T.I., University of Vigo, Pontevedra, Spain) for HRMS analysis, the Centro de Materiais da Universidade do Porto (CEMUP, Porto, Portugal) for HRMS, and the Departamento de Quimica da Universidade de Aveiro (Portuguese NMR network) for the NMR analysis