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Immunofluorescent staining of α-tubulin in (a and b) apoptotic and (c and d) non-apoptotic HL-60 cells. Cells were fixed and stained with α-tubulin monoclonal antibody and fluorescein-conjugated secondary antibodies, as described in material and methods. Nuclear morphology in the same subset which signifies the stage of apoptosis is clarified using Hoechst staining on the same cells (c and d). A, apoptotic; N, non-apoptotic; D, differentiated.
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Microtubules (MT) are important components of cell cytoskeleton and play key roles in cell motility mitosis and meiosis. They are also the targets of several anticancer agents which indicating their importance in maintaining cell viability. Microtubular reorganization contributing to apoptotic morphology occurs in normal and neoplastic cells underg...
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... assay, so those cells were at the latest stage of apoptosis. Examination of α-tubulin-stained cytocentrifuge preparations showed that all cells both treated and untreated showed some tubulin positivities. In apoptotic cells, two patterns of MT were observed: lack of staining and faint, disperse pattern of staining. No obvious MTOC was observe (Fig. 4 a and b). In normal cells, half displayed polymerized tubulin structures in cytoplasm and a Faint microtubular network were seen on or around the nucleus (Fig. 4 c and d). Immunofluorescence staining for the α-acetylated MT showed changes comparable to that of α-tubulin, but there was no consistent staining of α-acetylated cell in apoptotic ...
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... untreated showed some tubulin positivities. In apoptotic cells, two patterns of MT were observed: lack of staining and faint, disperse pattern of staining. No obvious MTOC was observe (Fig. 4 a and b). In normal cells, half displayed polymerized tubulin structures in cytoplasm and a Faint microtubular network were seen on or around the nucleus (Fig. 4 c and d). Immunofluorescence staining for the α-acetylated MT showed changes comparable to that of α-tubulin, but there was no consistent staining of α-acetylated cell in apoptotic cells (Fig. 5 a and b). In non-apoptotic cells, a fine rim of acetylated α-tubulin was seen around the nucleus (Fig. 5 c and ...
Citations
... Tubulin detyrosination occurs frequently in breast cancer and is linked to tumor aggressivity [24]. Reduced abundance of α and α-acetylated tubulin is associated with enhanced apoptosis in leukemia cells [32]. Acetylated α-tubulin is present in microtubules that under depolymerising condition are more stable than the majority of cytoplasmic microtubules [31,33]. ...
The proper folding pathway of α and β-tubulin into the α/β-tubulin heterodimers involve five Tubulin Binding Cofactors (TBCA to TBCE). TBCC plays a crucial role in the formation of polymerization-competent the α/β-tubulin heterodimers. To evaluate the impact of microtubule mass and dynamics on the phenotype and chemosensitivity of breast cancer cells, we targeted TBCC in human breast adenocarcinoma and developed variants of breast cancer cells with modified content of TBCC. We have shown that the modifications in TBCC expression level influenced tubulin fraction distribution and microtubule dynamics. Cell cycle distribution and the durations of mitosis and S-phase were altered. The proliferation rate in vitro was slightly modified whereas in vivo the TBCC variants presented major differences in tumor growth capacity. Chemosensitivity to antimicrotubule agents (paclitaxel and vinorelbine) as well as to gemcitabine was observed to be dependent on the cell cycle distribution of the TBCC variants. These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype, cell cycle progression and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterization of resistant phenotypes.
... Tubulin detyrosination occurs frequently in breast cancer and is linked to tumor aggressivity [24]. Reduced abundance of α and α-acetylated tubulin is associated with enhanced apoptosis in leukemia cells [32]. Acetylated α-tubulin is present in microtubules that under depolymerising condition are more stable than the majority of cytoplasmic microtubules [31,33]. ...
Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of alpha and beta tubulins into polymerization-competent tubulin heterodimers.
We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells.
TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts.
These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.
This study was carried out to investigate the effects of 100 and 217 Hz extremely low-frequency pulsed electromagnetic fields (ELF-PEMF) on cell proliferation, actin reorganization, and ROS generation in a human breast carcinoma cells (T47D). Cells were exposed for 24-72 h, at 100 and 217 Hz, 0.1 mT. The treatment induced a time dependent decrease in cell growth after 72 h and revealed an increase in fluorescence intensity in cytoplasm and actin aggregations around the nucleus as detected by fluorescence microscopy. The amount of actin in T47D cells increased after 48 h exposure to 100 Hz and 24 h to 217 Hz while no changes in nuclear morphology were detected. Exposing the cells to 217 Hz for 72 h caused a dramatically increase of intracellular ROS generation while with exposure to 100 Hz it remained nearly unchanged. These results suggest that exposure to ELF-PEMF (100, 217 Hz, 0.1 mT) are able inducing an increase of actin level, its migration toward nucleus but despite of these changes and dramatically increase in ROS generation the symptoms of apoptosis were not observed. Our results support the hypothesis that cell response to EMF may only be observed at certain window effects; such as frequency and intensity of EMF parameters.
