Is the Fate of Clinical Candidate Arry-520 Already Sealed? Predicting Resistance in Eg5–Inhibitor Complexes

Arry-520 is an advanced drug candidate from the Eg5 inhibitor class undergoing clinical evaluation in patients with relapsed or refractory multiple myeloma. Here we show by structural analysis that Arry-520 binds stoichiometrically to the motor domain of Eg5 in the conventional allosteric loop L5 pocket in a complex that suggests the same structural mechanism as other Eg5 inhibitors. We have previously shown that acquired resistance through mutations in the allosteric binding site located at loop L5 in the Eg5 structure appears to be independent of the inhibitors' scaffold, which suggests that Arry-520 will ultimately have the same fate. When Arry-520 was assessed in two cell lines selected for the expression of either Eg5(D130A) or Eg5(L214A) STLC-resistant alleles, mutations previously shown to convey resistance to this class of inhibitors, it was inactive in both. Surprisingly, when the cells were challenged with ispinesib, another Eg5 inhibitor, the Eg5(D130A) cells were resistant, but those expressing Eg5(L214A) were strikingly sensitive. Molecular dynamics simulations suggest that subtle differences in ligand binding and flexibility in both compound and protein may alter allosteric transmission from the loop L5 site that do not necessarily result in reduced inhibitory activity in mutated Eg5 structures. Whilst we predict that cells challenged with Arry-520 in the clinical setting are likely to acquire resistance through point mutations in the Eg5 binding site, the data for ispinesib suggests that this resistance mechanism is not scaffold independent as previously thought, and new inhibitors can be designed that retain inhibitory activity in these resistant cells

Impacto do programa bolsa família sobre a frequência escolar: o caso da agricultura familiar no Nordeste do Brasil.

O objetivo deste trabalho é avaliar o impacto do programa de transferência de renda condicionada Bolsa Família sobre a frequência escolar de crianças e adolescentes de cinco a 14 anos na agricultura familiar dos estados de Pernambuco, Ceará, Sergipe e Paraíba. Nessa investigação, o trabalho faz uso de dados primários (pesquisa de campo) e dados secundários (PNAD, 2005) para obter estimativas de propensity score. Os resultados indicam que, de forma geral, o programa eleva a frequência escolar das referidas crianças no intervalo de 5,4 a 5,9 pontos percentuais. Contudo, há importantes diferenças quando se considera meninas e meninos separadamente, sendo o programa eficaz no primeiro caso e ineficaz no segundo. Ou seja, apesar da avaliação positiva para as meninas, não parece haver efeito do programa sobre a frequência escolar dos meninos, o que pode estar associado a diferenças de gênero nos custos de oportunidades do investimento em capital humano no meio rural

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

The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

Background Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro. Methods Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed. Results Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment. Conclusions These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma

The decatenation checkpoint

The decatenation checkpoint delays entry into mitosis until the chromosomes have been disentangled. Deficiency in or bypass of the decatenation checkpoint can cause chromosome breakage and nondisjunction during mitosis, which results in aneuploidy and chromosome rearrangements in the daughter cells. A deficiency in the decatenation checkpoint has been reported in lung and bladder cancer cell lines and may contribute to the accumulation of chromosome aberrations that commonly occur during tumour progression. A checkpoint deficiency has also been documented in cultured stem and progenitor cells, and cancer stem cells are likely to be derived from stem and progenitor cells that lack an effective decatenation checkpoint. An inefficient decatenation checkpoint is likely to be a source of the chromosome aberrations that are common features of most tumours, but an inefficient decatenation checkpoint in cancer stem cells could also provide a potential target for chemotherapy

PP2A/B55 and Fcp1 regulate Greatwall and Ensa desphorylation during mitotic exit

Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, neither Fcp1 nor PP2A appear to essential to dephosphorylate the bulk of mitotic Cdk1 substrates following Cdk1 inhibition. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit

Expression of survivin detected by immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of leiomyosarcoma and synovial sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Survivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma.</p> <p>Methods</p> <p>Tumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS).</p> <p>Results</p> <p>We detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Cox's regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus.</p> <p>Conclusion</p> <p>Survivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.</p

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

<p>Abstract</p> <p>Background</p> <p>Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.</p> <p>Methods</p> <p>The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied <it>in vitro </it>by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.</p> <p>Results</p> <p>Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.</p> <p>Conclusions</p> <p>The study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.</p

Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis.Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell.Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the transcriptional activity of AIB1