THE EFFECTS OF ISOPROPYL N-PHENYL CARBAMATE ON THE GREEN ALGA OEDOGONIUM CARDIACUM : I. Cell Division

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2°C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10-4 M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-µm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH

Cellular Senescence: Ex Vivo p53-Dependent Asymmetric Cell Kinetics

Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture

Reproducibility Project: Cancer Biology

The Reproducibility Project: Cancer Biology was an initiative to independently replicate selected experiments from a number of high-profile papers in the field of cancer biology. In the end 50 experiments from 23 papers were repeated. The final two outputs from the project recount in detail the challenges the project team encountered while repeating these experiments ('Challenges for assessing replicability in preclinical cancer biology': https://elifesciences.org/articles/67995), and report the results of a meta-analysis that combined the results from all the experiments ('Investigating the replicability of preclinical cancer biology': https://elifesciences.org/articles/71601). The project was a collaboration between the Center for Open Science and Science Exchange with all papers published by eLife