Bernard Ducommun

Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B–Cdk1.

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro‐centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule‐nucleating activity of the mtoc 1. Several microtubule‐associated proteins have been characterized by their ability to bind taxol‐stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.

Variation of the immunolabelling of the α1-isotubulin in the mitotic spindle ofPhysarum polycephalum

SummaryImmunofluorescent labelling ofPhysarum microtubules with a new antibody specific for the α1-isotubulin has been compared with the labelling with an antibody specific for β-isotubulins and an antibody with recognizes tubulin chains terminated by an aromatic amino-acid. In agreement with the known presence of only one α-isotype in amoebae and several α-isotypes in plasmodia, the immunofluorescence of the mitotic spindle was qualitatively identical, but lower in plasmodia than in amoebae. In all cases except one, there were no relative variations of immuno-fluorescence staining with the three antibodies, from metaphase to telophase, in spindles sampled. In plasmodia grown at optimal temperature, both during normal or perturbed mitosis, the immunostaining of the α1isotype decreased sharply after metaphase, while the staining obtained with the two other antibodies did not vary significantly. The immunologic determination of the relative amount of the α1-isotubulin in the tubulin pool and in isolated mitotic microtubules could not account for this observation.

Effect of phenylarsine oxide on the fission yeast Schizosaccharomyces pombe cell cycle

Phosphotyrosyl turnover is an essential regulatory mechanism for many biological processes, and the balance between tyrosine kinases and phosphatases plays a major role in the control of cell proliferation. Phenylarsine oxide (PAO), a potent inhibitor of tyrosine phosphatases (PTPase), was used to investigate the involvement of PTPase in the growth and control of the cell cycle of the fission yeast Schizosaccharomyces pombe. Cell proliferation was arrested by treatment with PAO, which was found to inhibit cdc25 PTPase in vitro but appeared not to act in vivo on this mitosis inducer. The PAO-treated cells displayed a mono- or binucleated phenotype and a DNA content that was either 2C or 4C, indicating a cell cycle arrest with a failure to complete cytokinesis. Entry into the cell division cycle from the G0 quiescent stage was also delayed by treatment with PAO. These results suggest that a number of key events in the mitotic cell cycle are regulated by as yet unidentified PTPases.

Hyperspectral polarized light scattering to study tumor cells in in-vitro samples

Supercontinuum laser sources provide a very useful tool to characterize materials and scattering media such as nanomaterials in suspensions, aerosols or paint coatings. Onera, The French Aerospace Lab, has developed a fast, in-line and comprehensive optical characterization method. The hyperspectral polarized angular light measurements by tumor cells exhibit a unique signature. We propose an original way to probe tumor cells in in-vitro samples. First experimental results are presented with potential applications. This is the first time in our knowledge that hyperspectral, polarimetric and angular signature of MCTS is reported.

Reversible growth arrest of 3D tumor spheroids stored in oxygen absorber-induced anoxia

Multicellular tumor spheroids models are of increasing interest in preclinical studies and pharmacological evaluation. However, their storage and transport is often a limitation because it requires adapted and expensive procedures. Here, we propose a very simple method to store 3D spheroids, using a procedure based on oxygen absorber-induced anoxia. We report that oxygen absorbers allow generating an anoxic environment for spheroid storage in culture plates. Oxygen absorber-induced anoxia fully and reversibly arrests spheroid growth for 4 days at 37°C and up to 18 days at 4°C. We then show that the response to etoposide is comparable in spheroids preserved in conditions of absorber-induced anoxia at 4°C and spheroids kept in normoxia at 37°C. These results represent a major improvement that should simplify the storage, transport and use of 3D spheroids.

Evaluation by quantitative image analysis of anticancer drug activity on multicellular spheroids grown in 3D matrices

Pharmacological evaluation of anticancer drugs using 3D in vitro models provides invaluable information for predicting in vivo activity. Artificial matrices are currently available that scale up and increase the power of such 3D models. The aim of the present study was to propose an efficient and robust imaging and analysis pipeline to assess with quantitative parameters the efficacy of a particular cytotoxic drug. HCT116 colorectal adenocarcinoma tumor cell multispheres were grown in a 3D physiological hyaluronic acid matrix. 3D microscopy was performed with structured illumination, whereas image processing and feature extraction were performed with custom analysis tools. This procedure makes it possible to automatically detect spheres in a large volume of matrix in 96-well plates. It was used to evaluate drug efficacy in HCT116 spheres treated with different concentrations of topotecan, a DNA topoisomerase inhibitor. Following automatic detection and quantification, changes in cluster size distribution with a topotecan concentration-dependent increase of small clusters according to drug cytotoxicity were observed. Quantitative image analysis is thus an effective means to evaluate and quantify the cytotoxic and cytostatic activities of anticancer drugs on 3D multicellular models grown in a physiological matrix.

Optical signature of multicellular tumor spheroid using index-mismatch-induced spherical aberrations

The development of new cancer treatments and the early prediction of their therapeutic potential are often made difficult by the lack of predictive pharmacological models. The 3D multicellular tumor spheroid (MCTS) model offers a level of complexity that recapitulates the three-dimensional organization of a tumor and appears to be fairly predictive of therapeutic efficiency. The use of spheroids in large-scale automated screening was recently reported to link the power of a high throughput analysis to the predictability of a 3D cell model. The spheroid has a radial symmetry; this simple geometry allows establishing a direct correlation between structure and function. The outmost layers of MCTS are composed of proliferating cells and form structurally uniform domain with an approximate thickness of 100 microns. The innermost layers are composed of quiescent cells. Finally, cells in the center of the spheroid can form a necrotic core. This latest region is structurally heterogeneous and is poorly characterized. These features make the spheroid a model of choice and a paradigm to study the optical properties of various epithelial tissues. In this study, we used an in-vitro optical technique for label-free characterization of multicellular systems based on the index- mismatch induced spherical aberrations. We achieve to monitor and characterize the optical properties of MCTS. This new and original approach might be of major interest for the development of innovative screening strategies dedicated to the identification of anticancer drugs.

Abstract 560: Mechanical stress activates a mitotic checkpoint in multicellular tumor spheroids.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Expanding solid tumors are subjected to mechanical stress that impact on their growth rate and development. However little is known on the mechanisms by which mechanical cues are acting on tumor cell biology. To address that issue, we used MultiCellular Tumor Spheroid (MCTS), a 3D model recapitulating the microenvironment, the proliferative gradient and cell-cell interactions found in a tumor. To test the impact of mechanical stress on tumor cell proliferation, we first designed, produced and used dedicated polymer microdevices in which MCTS engineered to express fluorescent biomarkers were confined to apply mechanical constraints. We observe that under constraints, MCTS display a high proportion of mitotic cells in low proliferative regions of confined spheroids. We show that these cells are being arrested in mitosis for at least 24 hours (EdU incorporation neg.) and that mitotic arrest is not caused by impairment of rounding. We next used live SPIM (Selective Plane Illumination Microscopy) 3D imaging to monitor mitosis progression in isotropically constrained MCTS. We show that constraint impairs bipolar spindle assembly and delays progression toward metaphase-anaphase transition. Our data indicate that in a multicellular structure mechanical constraints are responsible for a defect in cell cycle progression associated with a mitotic arrest. Citation Format: Annaick Desmaison, Katia Grenier, Celine Frongia, Corinne Lorenzo, Bernard Ducommun, Valerie Lobjois. Mechanical stress activates a mitotic checkpoint in multicellular tumor spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 560. doi:10.1158/1538-7445.AM2013-560