Mitotic Furrow Research Articles

Abstract 5156: In vitro application of tumor-treating fields to suppress tunneling nanotubes in mesothelioma

Abstract The purpose of this study was to evaluate the effects of tumor-treating fields (TTFields) on tunneling nanotubes (TNTs) in malignant mesothelioma. TTFields have emerged as a therapeutic modality for the treatment of glioblastoma, through application of alternating electric fields, which exert dipole alignment and dielectrophoretic force. The dielectrophoretic force is intensified in cellular compartments that are non-uniform in size and shape such as the mitotic furrow. TNTs are filamentous actin protrusions that are conduits for intercellular communication and transport of vital cell signals that stimulate cell growth, invasion, and resistance to treatment. TNTs are highly prevalent in malignant pleural mesothelioma. We hypothesized that by creating dielectrophoretic force, TTFields may disrupt or prevent the formation of mesothelioma TNTs and thus sensitize these cells to the cytotoxic effect of chemotherapy and TTFields. TTFields (1.1-1.6 V/cm; 150-200 kHz) were applied using the inovitro system to VAMT and MSTO malignant mesothelioma cell lines. TNT index (average # of TNTs/cell) was determined at 24, 48, and 72 hours of TTFields application. Cell viability assessment using evaluation of cell count and colony formation assays were performed to determine effects of TTFields either alone or in combination with varying concentrations of cisplatin (range 0-10 µM) or pemetrexed (0-512 nM). In separate experiments, we examined the effects of cell cycle inhibition on TNTs using the compound AZD 5438 (Tocris Biosciences). Application of continuous TTFields suppressed TNT formation by 60% over a 72-hour period. This suppression was achieved by the 24-hour timepoint, and maintained over the 72 hours. Assessment of cell viability showed high rate of cell death at the stated frequencies and intensities. The addition of cisplatin or pemetrexed to TTFields application significantly decreased the cell count. The combination of TTFields and low concentration cisplatin (0.1-1 µM) resulted in enhanced treatment efficacy as evaluated using the colony formation assay. Complete inhibition of the cell cycle paradoxically stimulated a significantly higher number of TNTs. Here, we show that TTFields application suppresses TNTs formation in malignant mesothelioma cell lines. Treatment with TTFields leads to enhanced treatment efficacy when combined with standard of care chemotherapeutic drugs cisplatin and pemetrexed. Additional optimization of the applied TTFields intensities and frequencies may further improve treatment efficacy. The sharp rise in TNTs seen following cell cycle inhibition is a potential cellular stress response to cancer-directed treatment. Suppression of TNT formation, and thus TNT-mediated intercellular communication networks in tumors, is a potential mechanism of TTF efficacy in treatment of mesothelioma, glioblastoma, and other invasive cancers. Citation Format: Akshat Sarkari, Michal Munster, Einav Zeevi, Moshe Giladi, Emil Lou. In vitro application of tumor-treating fields to suppress tunneling nanotubes in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5156.

Tumour-treating fields (TTFields): Investigations on the mechanism of action by electromagnetic exposure of cells in telophase/cytokinesis

Tumour-treating fields (TTFields) use alternating electric fields which interfere with dividing cells, thereby reducing tumour growth. Previous reports suggest that electrical forces on cell structure proteins interfered with the chromosome separation during mitosis and induced apoptosis. In the present report we evaluate electromagnetic exposure of cells in telophase/cytokinesis in order to further analyse the mechanism of action on cells. We performed numerical electromagnetic simulations to analyse the field distribution in a cell during different mitotic phases. Based thereon, we developed an electric lumped element model of the mitotic cell. Both the electromagnetic simulation and the lumped element model predict a local increase of the specific absorption rate (SAR) as a measure of the electromagnetically induced power absorption density at the mitotic furrow which may help to explain the anti-proliferative effect. In accordance with other reports, cell culture experiments confirmed that TTFields reduce the proliferation of different glioma cell lines in a field strength- and frequency-dependent manner. Furthermore, we found an additional dependence on the commutation time of the electrical fields. The report gives new insights into TTFields’ anti-proliferative effect on tumours, which could help to improve future TTFields application systems.

ET-68 * ALTERNATING ELECTRIC FIELDS PERTURB THE LOCALIZATION OF CYTOKINETIC FURROW PROTEINS AND CAUSE ABERRANT MITOTIC EXIT

The NovoTTF-100A is an FDA approved device for the treatment of recurrent Glioblastoma. However, the precise nature of its effects on cells that support its clinical efficacy is poorly understood. We have found that the alternating electric fields generated by the NovoTTF-100A (TTFields) disrupt cells during the metaphase to anaphase transition. Cells exposed to TTFields during mitosis appear normal within metaphase, but exhibit prolonged anaphase accompanied by random and uncontrolled membrane contractions referred to as mitotic blebbing arising from dysregulation of contractile elements normally localized to the cytokinetic furrow. This phenomenon leads to aberrant mitotic exit, leading to decreased proliferation and increased apoptosis that is influenced by p53 mutational status. Further analysis demonstrates that treated cells exhibited features of so-called immunogenic cell death capable of stimulating immune responses deranged cells. The ability of alternating electric fields to thus affect cellular physiology is dependent on their ability to perturb the function of specific proteins that possess high dipole moments. The Septin heterotrimer is composed of Septins 2, 6 and 7 and has a high dipole moment of 2711 Debyes. This Septin complex is essential for the organization and the regulation of cytokinetic furrow contraction which results in daughter cell formation. Importantly, perturbation of Septins or their binding partners is reported to result in mitotic catastrophe similar to that observed during TTFields exposure. We report that TTFields perturb normal Septin localization during both mitosis and cell spreading demonstrating a direct effect of TTFields on a protein involved in mitosis. Together, these data strongly support a model that explains how TTFields interfere with normal mitotic progression by disrupting mitotic furrow regulation causing mitotic catastrophe and aberrant mitotic exit. This leads to a form of immunogenic cell death that may sufficiently reduce intratumoral immune privilege to allow for tumor elimination in some patients.

A mitotic kinesin-6, Pav-KLP, mediates interdependent cortical reorganization and spindle dynamics in Drosophila embryos

Introduction Dynamic interactions between the mitotic spindle, the cell cortex and the plasma membrane underlie many crucial cellular phenomena. For example, during cell division, the localization of the cleavage furrow is specified by the mitotic spindle (Glotzer, 2001; Scholey et al., 2003; Odell and Foe, 2008), whereas actomyosin-based cortical contractions contribute to spindle morphogenesis (Rosenblatt et al., 2004). In addition, membrane transport to the furrow is crucial for cell-cell abscission and the completion of cytokinesis (Finger and White, 2002). Recently, it has been shown that vesicles associated with F-actin are transported on furrow microtubules (MTs) (Albertson et al., 2008). However, the underlying molecular mechanisms remain largely obscure. Drosophila embryogenesis is amenable to the study of interactions between the spindle, cortex and membrane using genetic analysis, inhibitor microinjection and microscopy (Glover, 2005). In the Drosophila syncytial embryo, nuclei share the same cytoplasm and undergo 13 synchronous divisions without any intervening cytokinesis; however, following the migration of the nuclei to the embryonic surface during cycle 9, mitotic furrows form transiently around each spindle (Mazumdar and Mazumdar, 2002). The mitotic furrows grow longer during each metaphase, serving as barriers to maintain the spacing between adjacent spindles, and then regress during late anaphase and telophase. Subsequently, during interphase of cycle 14, cellularization furrows descend from the blastoderm surface to partition the nuclei into individual cells. Many studies suggest that spindle morphogenesis, as monitored by the separation of the spindle poles, depends upon a balance of forces generated by multiple molecular motors that guide the spindle through a sequence of quiescent steady states interspersed with phases of rapid spindle-pole separation (Sharp et al., 2000; Brust-Mascher et al., 2004; Cytrynbaum et al., 2005). The mitotic furrows maintain a constant distance to the poles throughout mitosis, and perturbations of mitotic molecules can cause both spindle and cortical defects during the early mitotic cycles (Cytrynbaum et al., 2005). To test the hypothesis that this force balance reflects a dynamic relationship between the spindle and the cortex, we need to know the identity of the crucial molecules that influence the structure and dynamics of both the spindle and the cortex. It is unknown how the spindle affects cortical reorganization and, conversely, how the cortex affects spindle morphogenesis. Both cellularization and metaphase furrow formation in the early Drosophila embryo are driven by the MT-dependent delivery of membrane vesicles to the furrow region (Strickland and Burgess, 2004; Riggs et al., 2003). As in conventional cytokinesis, new furrow membrane does not seem to be derived from the expansion of the pre-existing surface membrane, but rather forms through the insertion of membrane from internal stores, such as the recycling endosome (RE), Golgi or both (Lecuit and Weichaus, 2000; Sisson A mitotic kinesin-6, Pav-KLP, mediates interdependent cortical reorganization and spindle dynamics in Drosophila embryos

SpH‐protease exists in Caco‐2 cells

We have described a 60 kDa nuclear cysteine protease that degrades sperm histones during male chromatin remodeling after fertilization in sea urchins (SpH protease). This enzyme was shown to be localized in the mitotic furrow and to be involved in cell cycle control in sea urchin embryos. The N terminal sequence and the gene encoding this protease have homology with human cathepsin L family; therefore we investigated with antibodies raised against the sea urchins protease if a similar protease is present in Caco‐2 cells. Among Caco 2 chromosomal proteins obtained from confluent cell cultures, we found by Western blots, two bands migrating at 60 and 100 kDa in SDS/PAGE. We have also used these anti SpH protease antibodies to immunolocalize this protease in the intracellular context. This protease in mitotic cells localize on the mitotic furrow and remains in the nucleus in binuclear cells. On the other hand, when inhibitors of cysteine‐proteasas were used in different phases of the cell cycle we observed a disorganization of the mitotic spindle and an alteration in the localization of the protease during the S phase affecting DNA replication. Our results demonstrate that in Caco 2 cells exist a variant of cathepsin L protease that exhibits a similar localization to that was described for embryos of sea urchin, indicating a putative rol in cell proliferation. FONDECYT 11070067.

Centrosomes and the Scrambled protein coordinate microtubule-independent actin reorganization.

In Drosophila syncytial blastoderm embryos, centrosomes specify the position of actin-based interphase caps and mitotic furrows. Mutations in the scrambled locus prevent assembly of mitotic furrows, but do not block actin cap formation. The scrambled gene encodes a protein that localizes to the mitotic furrows and centrosomes. Sced localization, actin reorganization from caps into mitotic furrows, and centrosome-coordinated assembly of actin caps are not blocked by microtubule disruption. Our results indicate that centrosomes may coordinate assembly of cortical actin caps through a microtubule-independent mechanism, and that Scrambled mediates a second microtubule-independent process that drives mitotic furrow assembly.

Orientation of mitosis of fibroblasts is determined in the interphase.

Time-lapse films of the cultures of mouse L fibroblasts have been analyzed to find out the angles between the direction of the big axis of interphase nucleus before mitosis and the direction of the mitotic furrow in the same cell. In 41 out of 51 mitoses this angle has been found to be more than 60 degrees . Thus, the orientation of mitotic furrows depends on the internal polarity of the cell before mitosis.