The mechanical force between cells and the extracellular microenvironment is crucial to many physiological processes such as cancer metastasis and stem cell differentiation. Mitosis plays an essential role in all thes...The mechanical force between cells and the extracellular microenvironment is crucial to many physiological processes such as cancer metastasis and stem cell differentiation. Mitosis plays an essential role in all these processes and thus an in-depth understanding of forces during mitosis gains insight into disease diagnosis and disease treatment. Here, we develop a traction force microscope method based on monolayer fluorescent beads for measuring the weak traction force (tens of Pa) of mitotic cells in three dimensions. We quantify traction forces of human ovarian granulosa (KGN) cells exerted on the extracellular matrix throughout the entire cell cycle in three dimensions. Our measurements reveal how forces vary during the cell cycle, especially during cell division. Furthermore, we study the effect of paclitaxel (PTX) and nocodazole (NDZ) on mitotic KGN cells through the measurement of traction forces. Our results show that mitotic cells with high concentrations of PTX exert a larger force than those with high concentrations of NDZ, which proved to be caused by changes in the structure and number of microtubules. These findings reveal the key functions of microtubule in generating traction forces during cell mitosis and explain how dividing cells regulate themselves in response to anti-mitosis drugs. This work provides a powerful tool for investigating cell-matrix interactions during mitosis and may offer a potential way to new therapies for cancer.展开更多
Metaphase-arrest agents and hyperthermia are both known to be capable of inducing apoptosis, and they have been used, separately, in cancer treatments. Here, we have examined whether the two treatments together may ha...Metaphase-arrest agents and hyperthermia are both known to be capable of inducing apoptosis, and they have been used, separately, in cancer treatments. Here, we have examined whether the two treatments together may have a synergistic effect. We find that when H-HeLa cells are arrested in metaphase with spindle poisons (nocodazole or paclitaxel) and then subjected to mild heat treatment (41.5℃), they exhibit morphological changes typical of apoptosis within three hours. Moreover, those changes are blocked by the pan-caspase inhibitor zVAD-fmk, indicating apoptosis, and activated Procaspase 3 is detected by immunoblotting and by staining with the fluoresce-in-labelled caspase inhibitor FAM-VAD-fmk. Interphase cells treated in the same way do not under-go apoptosis, even with spindle poisons present. Induction of apoptosis is more rapid when the cells have been arrested longer in metaphase, suggesting that accumulation or depletion of some cellular component(s) during metaphase-arrest may make them more susceptible to hyperthermia. Further work is in progress to test whether other cell lines exhibit the same behavior and to learn more about the mechanism. The phenomenon is of interest because it may provide clues to how hyperthermia induces cell death and may yield novel therapeutic approaches to block or stimulate apoptosis.展开更多
Wallerian degeneration,the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury,is essential for creating a permissive microenvironment for nerve regeneration,and involves cy...Wallerian degeneration,the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury,is essential for creating a permissive microenvironment for nerve regeneration,and involves cytoskeletal reconstruction.However,it is unclear whether microtubule dynamics play a role in this process.To address this,we treated cultured sciatic nerve explants,an in vitro model of Wallerian degeneration,with the microtubule-targeting agents paclitaxel and nocodazole.We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation,whereas nocodazole-induced microtubule destabilization inhibited these processes.Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration,as well as functional recovery of nerve conduction and target muscle and motor behavior,respectively.These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration.This study was approved by the Animal Care and Use Committee of Southern Medical University,China(approval No.SMUL2015081) on October 15,2015.展开更多
AIM： To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 （DR5） and apoptosis. METHODS： Anchorage-dependent EC9706 cells of esophagea...AIM： To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 （DR5） and apoptosis. METHODS： Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS： Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent ceils. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION： Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.展开更多
Objective：Microtubule damaging agents such as nocodazole have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of apoptosis induction activity of these agents are still unclear. We pre...Objective：Microtubule damaging agents such as nocodazole have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of apoptosis induction activity of these agents are still unclear. We previously demonstrated that nocodazole induced apoptosis in human leukemic HL-60 cells. In order to investigate the genes involved in this process, cDNA RDA was performed to identify upregulated genes in the apoptosis process induced by nocodazole. Methods ：Sixteen up-regulated genes were identified and confirmed to be differentially expressed, including ribosomal proteins, cytochrome b, Wilm＇s tumor related protein QM gene, alpha-tubulin isoform 1, heterogeneous nuclear ribonucleoprotein C enzymes, follicular lymphoma variant translocation 1, Ribophorin I , etc. Results：It was the first time for several genes reported to be related to apoptosis, including one functionally unkown gene, BING4. We also analyzed the expression of these genes in apoptosis induced by etoposide, and results showed 9 genes were also up-regulated, indicating they could be important genes involved in apoptosis. Conclusion：The study provides important clues to understand mechanism of apoptosis induced by nocodazole, and establishes foundation for further study.展开更多
基金The authors gratefully acknowledge financial support from the National Natural Science Foundation of China(Nos.11872355,11627803,12072339,and 11872354)the Strategic Priority Research Program of the Chinese Academy of Science(No.XDB22040502).
文摘The mechanical force between cells and the extracellular microenvironment is crucial to many physiological processes such as cancer metastasis and stem cell differentiation. Mitosis plays an essential role in all these processes and thus an in-depth understanding of forces during mitosis gains insight into disease diagnosis and disease treatment. Here, we develop a traction force microscope method based on monolayer fluorescent beads for measuring the weak traction force (tens of Pa) of mitotic cells in three dimensions. We quantify traction forces of human ovarian granulosa (KGN) cells exerted on the extracellular matrix throughout the entire cell cycle in three dimensions. Our measurements reveal how forces vary during the cell cycle, especially during cell division. Furthermore, we study the effect of paclitaxel (PTX) and nocodazole (NDZ) on mitotic KGN cells through the measurement of traction forces. Our results show that mitotic cells with high concentrations of PTX exert a larger force than those with high concentrations of NDZ, which proved to be caused by changes in the structure and number of microtubules. These findings reveal the key functions of microtubule in generating traction forces during cell mitosis and explain how dividing cells regulate themselves in response to anti-mitosis drugs. This work provides a powerful tool for investigating cell-matrix interactions during mitosis and may offer a potential way to new therapies for cancer.
文摘Metaphase-arrest agents and hyperthermia are both known to be capable of inducing apoptosis, and they have been used, separately, in cancer treatments. Here, we have examined whether the two treatments together may have a synergistic effect. We find that when H-HeLa cells are arrested in metaphase with spindle poisons (nocodazole or paclitaxel) and then subjected to mild heat treatment (41.5℃), they exhibit morphological changes typical of apoptosis within three hours. Moreover, those changes are blocked by the pan-caspase inhibitor zVAD-fmk, indicating apoptosis, and activated Procaspase 3 is detected by immunoblotting and by staining with the fluoresce-in-labelled caspase inhibitor FAM-VAD-fmk. Interphase cells treated in the same way do not under-go apoptosis, even with spindle poisons present. Induction of apoptosis is more rapid when the cells have been arrested longer in metaphase, suggesting that accumulation or depletion of some cellular component(s) during metaphase-arrest may make them more susceptible to hyperthermia. Further work is in progress to test whether other cell lines exhibit the same behavior and to learn more about the mechanism. The phenomenon is of interest because it may provide clues to how hyperthermia induces cell death and may yield novel therapeutic approaches to block or stimulate apoptosis.
基金supported by the National Natural Science Foundation of China,Nos.82071386 (to JS),81870982 (to JS)&81571182 (to JS)the National Key Basic Research Program of China,No.2014CB542202 (to JS)+3 种基金the Program for Changjiang Scholars and Innovative Research Team in University of China,No.IRT-16R37 (to JS)Key Research&Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory of China,No.2018GZR110104008 (to HZ)Research Grant of Guangdong Province Key Laboratory of Psychiatric Disorders of China,No.N201904 (to JS)Natural Science Foundation of Guangdong Province of China,No.2017A030312009 (to JS)。
文摘Wallerian degeneration,the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury,is essential for creating a permissive microenvironment for nerve regeneration,and involves cytoskeletal reconstruction.However,it is unclear whether microtubule dynamics play a role in this process.To address this,we treated cultured sciatic nerve explants,an in vitro model of Wallerian degeneration,with the microtubule-targeting agents paclitaxel and nocodazole.We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation,whereas nocodazole-induced microtubule destabilization inhibited these processes.Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration,as well as functional recovery of nerve conduction and target muscle and motor behavior,respectively.These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration.This study was approved by the Animal Care and Use Committee of Southern Medical University,China(approval No.SMUL2015081) on October 15,2015.
基金Supported by National Natural Science Foundation of China,No. 30571697Outstanding Individual Innovation Foundation of Henan Province,China,No.074200510014
文摘AIM： To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 （DR5） and apoptosis. METHODS： Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS： Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent ceils. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION： Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.
文摘Objective：Microtubule damaging agents such as nocodazole have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of apoptosis induction activity of these agents are still unclear. We previously demonstrated that nocodazole induced apoptosis in human leukemic HL-60 cells. In order to investigate the genes involved in this process, cDNA RDA was performed to identify upregulated genes in the apoptosis process induced by nocodazole. Methods ：Sixteen up-regulated genes were identified and confirmed to be differentially expressed, including ribosomal proteins, cytochrome b, Wilm＇s tumor related protein QM gene, alpha-tubulin isoform 1, heterogeneous nuclear ribonucleoprotein C enzymes, follicular lymphoma variant translocation 1, Ribophorin I , etc. Results：It was the first time for several genes reported to be related to apoptosis, including one functionally unkown gene, BING4. We also analyzed the expression of these genes in apoptosis induced by etoposide, and results showed 9 genes were also up-regulated, indicating they could be important genes involved in apoptosis. Conclusion：The study provides important clues to understand mechanism of apoptosis induced by nocodazole, and establishes foundation for further study.
