Structural centrosome aberrations promote non-cell-autonomous invasiveness

Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes

Structural centrosome aberrations favor proliferation by abrogating microtubule-dependent tissue integrity of breast epithelial mammospheres

Structural centrosome aberrations are frequently observed in early stage carcinomas, but their role in malignant transformation is poorly understood. Here, we examined the impact of overexpression of Ninein-like protein (Nlp) on the architecture of polarized epithelia in three-dimensional mammospheres. When Nlp was overexpressed to levels resembling those seen in human tumors, it formed striking centrosome-related bodies (CRBs), which sequestered Ninein and affected the kinetics of microtubule (MT) nucleation and release. In turn, the profound reorganization of the MT cytoskeleton resulted in mislocalization of several adhesion and junction proteins as well as the tumor suppressor Scribble, resulting in the disruption of epithelial polarity, cell-cell interactions and mammosphere architecture. Remarkably, cells harboring Nlp-CRBs displayed an enhanced proliferative response to epidermal growth factor. These results demonstrate that structural centrosome aberrations cause not only the disruption of epithelial polarity but also favor overproliferation, two phenotypes typically associated with human carcinomas.Oncogene advance online publication, 14 September 2015; doi:10.1038/onc.2015.332

Legislative Documents

Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents

Movie 4 related to fig2c: Basal extrusion of GFP-CEP131 expressing cell from MDCK 2D monolayer. from Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion

Movie shows brightfield signal (upper movie) and corresponding fluorescence signals (lower movie) representing mCardinal-ZO-1 (red) and GFP-CEP131 (green) (lower movie). MDCK were cells cultured in 2D monolayer, induced to express GFP-CEP131 for 48 hours and treated with etoposide to stimulate cell extrusion. Images were acquired every 20 minutes and movie displays 1 frame per second. Scale bars represent 10 microns

The 3' Untranslated Region of the Cyclin B mRNA Is Not Sufficient to Enhance the Synthesis of Cyclin B during a Mitotic Block in Human Cells

<div><p>Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR) of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents.</p> </div

Management of multiple myeloma in pregnancy: strategies for a rare challenge

Multiple myeloma is the second most commonly diagnosed hematologic malignancy. It is characterized by the accumulation of monoclonal plasma cells. It typically manifests in the sixth decade of life or later, whereas the incidence in patients who are younger than 40 years old is extremely rare. Here, we report the case of a 34-year-old prima gravida, diagnosed with a κ light-chain myeloma (Durie&amp;Salmon stage IIIA, International Staging System I) in the 23rd week of pregnancy. Our multimodal therapeutic approach during pregnancy, the delivery of a healthy male, and initiation of intensive anti-myeloma treatment thereafter (induction with bortezomib, cyclophosphamide, and dexamethasone, followed by tandem autologous peripheral blood stem cell transplantation) are described. Furthermore, we provide a comprehensive review of all 18 cases published between 1965 and 2010 in which a multiple myeloma was diagnosed and treated following different regimes and approaches before, during, or shortly after pregnancy. All delivered newborns were healthy, whereas the mothers' outcomes varied strongly. In our specific case, complete remission was achieved after tandem autologous peripheral blood stem cell transplantation. Emerging from these literature data and our case, we conclude that while awaiting delivery, the application of prednisolone as a nontoxic, but active anti-myeloma therapy can be recommended. Intensified postpartum anti-myeloma therapy should be induced as soon as possible to efficiently reduce myeloma burden and avoid organ damage in these young females

Degradation and synthesis of the double-chimeric cyclin B reporter throughout the cell cycle by live-cell imaging at the single-cell level.

<p>(A) Degradation of the reporter is reflected by a decrease in BG430 fluorescence intensity at the metaphase to anaphase transition following pulse-chase labeling. (B) Whole protein expression of the CYS reporter molecule is reflected by YFP fluorescence intensity throughout the cell cycle. (C) An overlay of YFP (green), BG430 (blue) and mCherry (red) fluorescence is depicted by the lower image series. (D) Whole protein expression, degradation and synthesis/accumulation of the CYS reporter and pulse-chase labeling with the SNAP substrate is depicted for the indicated cell cycle stages by overlaying of YFP and BG430 fluorescence. (E) YFP (green curve) and BG430 (blue curve) fluorescence intensity curves are shown and the corresponding cell cycle stages are indicated (see cell cycle stage between the dotted lines). Background fluorescence was subtracted and the initial fluorescence intensity level was set to 100%. (F) A curve displaying the synthesis rate/accumulation of the cyclin B reporter (as defined by the difference between YFP- and BG430-fluorescence) is indicated.</p

Characterization of cyclin B kinetics in the presence of the cyclin B 3’ UTR in human tumor- and hTERT-immortalized cells and in the presence of the microtubule-stabilizer taxol.

<p>YFP and BG430 fluorescence intensity curves as determined by live-cell imaging of U2Os, HeLa and hTERT RPE-1 cells expressing the CYS reporter with a long 3’ UTR sequence. HeLa and hTERT RPE-1 cells that are arrested in a mitotic block using a final nocodazole concentration of 200ng/mL in the growth medium are depicted in (A) and (B) (mean fluorescence intensities +/- SD are indicated, n=4). U2Os, HeLa and hTERT RPE-1 cells that are arrested in a mitotic block using a final taxol concentration in the growth medium of 1µM are depicted in (C, D and E) (mean fluorescence intensities +/- SD are indicated, n=4). For every shown experimental setting the analyzed cells are representatives of three independent experiments each of which included at least 50.000 cells.</p

A double-chimeric system allowing the study of cyclin B kinetics at the single cell level in the presence and absence of the cyclin B 3'UTR.

<p>(A) A double-chimeric cyclin B reporter protein allowed the monitoring of whole protein expression by YFP fluorescence and degradation by pulse-chase labeling with the membrane-permeable SNAP substrate BG430. (B) Exemplary protein kinetics of the double-chimeric cyclin B reporter with GFP-SNAP were assessed by Western Blotting. Expression of the reporter protein and endogenous cyclin B following a nocodazole release are shown. (C) Plasmid map of the retroviral pLNCX2 vector indicating the sequence encoding the double-chimeric reporter and the 3' untranslated region (UTR). (D) Listing of different 3'UTRs found in available cyclin B full-length clones. (E) Sequence of the full-length 3'UTR indicating the sequence of the short 3'UTR variant in red and the hexanucleotide sequences (AATAAA) and the CPEs (T_4-5A_1-2T) in bold.</p