69 results on '"Edward H. Cho"'
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2. Interleukin 10 and TNFα synergistically enhance the expression of the G protein-coupled formylpeptide receptor 2 in microglia
- Author
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Pablo Iribarren, Keqiang Chen, Wanghua Gong, Edward H. Cho, Stephen Lockett, Badarch Uranchimeg, and Ji Ming Wang
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Cytokines ,Chemotaxis ,Inflammation ,Protein Kinases ,Neuroimmunology ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Microglia are important participants in inflammatory responses in the central nervous system. We previously observed that tumor necrosis factor alpha (TNFα) induces the expression of the formylpeptide receptor mFPR2 on microglial cells. This chemoattractant receptor mediates microglial cell chemotaxis in response to a variety of peptides, including amyloid β peptide (Aβ42), a major pathogenic factor in Alzheimer’s disease (AD). In search for agents that regulate microglial activation, we unexpectedly found that IL-10 enhanced the expression of mFPR2 on TNFα-activated microglia. This was associated with a markedly increased microglial chemotaxis to Aβ42 and its endocytosis via mFPR2. Mechanistic studies revealed that the synergistic effect of IL-10 on TNFα-induction of mFPR2 in microglia was dependent on activation of p38 MAPK. Our results suggest that IL-10 may affect the pathogenic process of AD by up-regulating mFPR2 and thus favoring the recognition and internalization of Aβ42 by activated microglial cells.
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- 2007
- Full Text
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3. Supplementary Video 2 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Video 2 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
4. Supplementary Video 1 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Supplementary Video 1 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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- 2023
5. Supplementary Figure Legends 1-5 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Figure Legends 1-5 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
6. Supplementary Methods from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Supplementary Methods from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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- 2023
7. Supplementary Figure 1 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Figure 1 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
8. Supplementary Video 2 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Supplementary Video 2 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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- 2023
9. Supplementary Video 5 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Video 5 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
10. Supplementary Video 3 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Video 3 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
11. Supplementary Figure 5 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Figure 5 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
12. Supplementary Figure 3 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Figure 3 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
13. Supplementary Methods from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Methods from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
14. Data from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin β1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin β1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241–50]
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- 2023
15. Supplementary Video 1 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Video 1 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
16. Supplementary Figure 1 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
- Author
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Figure 1 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
17. Data from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulin-based protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated α-tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated α-tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread. [Cancer Res 2008;68(14):5678–88]
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- 2023
18. Supplementary Video 4 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Video 4 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
19. Supplementary Figure 3 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Figure 3 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
- Published
- 2023
20. Supplementary Figure Legends 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Supplementary Figure Legends 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
- Published
- 2023
21. Supplementary Figure 4 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
- Author
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Stuart S. Martin, Jing Yang, Kimberly C. Tuttle, Olga B. Ioffe, Jennifer R. Yoon, Michele I. Vitolo, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Rebecca A. Whipple
- Abstract
Supplementary Figure 4 from Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
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- 2023
22. Supplementary Figures 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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Jeffrey E. Green, Ann F. Chambers, Chand Khanna, Stephen Lockett, Edward H. Cho, Sylvain V. Costes, Zi-yao Liu, Mark J. Hoenorhoff, Anil K. Kamaraju, Holly Asmussen, Justin L. Simmons, Hynda Kleinman, and Dalit Barkan
- Abstract
Supplementary Figures 1-5 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton
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- 2023
23. Supplementary Figure 2 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Stuart S. Martin, Jennifer R. Yoon, Michael A. Matrone, Edward H. Cho, Eric M. Balzer, and Rebecca A. Whipple
- Abstract
Supplementary Figure 2 from Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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- 2023
24. AP-2α Regulates S-Phase and Is a Marker for Sensitivity to PI3K Inhibitor Buparlisib in Colon Cancer
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Mikhail V. Kulak, Anna C. Beck, Vivian W. Gu, Lily Paemka, Christopher M Franke, Tiandao Li, Shannon R. Landers, Matthew Gosse, Vincent T. Wu, Anthony J. Pamatmat, Kelsey E. Koch, Edward H. Cho, Ronald J. Weigel, Dakota T. Thompson, and Jeffrey R. White
- Subjects
0301 basic medicine ,Cancer Research ,Cell Survival ,Morpholines ,Buparlisib ,Aminopyridines ,medicine.disease_cause ,Article ,S Phase ,Small hairpin RNA ,Transcriptome ,03 medical and health sciences ,chemistry.chemical_compound ,Gene Knockout Techniques ,Mice ,0302 clinical medicine ,Cell Line, Tumor ,medicine ,Biomarkers, Tumor ,Animals ,Humans ,RNA-Seq ,Molecular Biology ,Protein kinase B ,PI3K/AKT/mTOR pathway ,Phosphoinositide-3 Kinase Inhibitors ,Gene Expression Profiling ,Cancer ,Cell cycle ,medicine.disease ,HCT116 Cells ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Oncology ,chemistry ,Transcription Factor AP-2 ,030220 oncology & carcinogenesis ,Colonic Neoplasms ,Cancer research ,RNA Interference ,Carcinogenesis - Abstract
Activating protein 2 alpha (AP-2α; encoded by TFAP2A) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize regulation of the transcriptome by AP-2α in colon cancer. CRISPR-Cas9 and short hairpin RNA were used to eliminate TFAP2A expression in HCT116 and a panel of colon cancer cell lines. AP-2α target genes were identified with RNA sequencing and chromatin immunoprecipitation sequencing. Effects on cell cycle were characterized in cells synchronized with aphidicolin and analyzed by FACS and Premo FUCCI. Effects on invasion and tumorigenesis were determined by invasion assay, growth of xenografts, and phosphorylated histone H3 (PHH3). Knockout of TFAP2A induced significant alterations in the transcriptome including repression of TGM2, identified as a primary gene target of AP-2α. Loss of AP-2α delayed progression through S-phase into G2–M and decreased phosphorylation of AKT, effects that were mediated through regulation of TGM2. Buparlisib (BKM120) repressed in vitro invasiveness of HCT116 and a panel of colon cancer cell lines; however, loss of AP-2α induced resistance to buparlisib. Similarly, buparlisib repressed PHH3 and growth of tumor xenografts and increased overall survival of tumor-bearing mice, whereas, loss of AP-2α induced resistance to the effect of PI3K inhibition. Loss of AP-2α in colon cancer leads to prolonged S-phase through altered activation of AKT leading to resistance to the PI3K inhibitor, Buparlisib. The findings demonstrate an important role for AP-2α in regulating progression through the cell cycle and indicates that AP-2α is a marker for response to PI3K inhibitors. Implications: AP-2α regulated cell cycle through the PI3K cascade and activation of AKT mediated through TGM2. AP-2α induced sensitivity to Buparlisib/BKM120, indicating that AP-2α is a biomarker predictive of response to PI3K inhibitors.
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- 2020
25. AP-2γ is Required for Maintenance of Pluripotent Mammary Stem Cells
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Nicholas Borcherding, Ronald J. Weigel, Vivian W. Gu, Victoria C. Cassady, Weizhou Zhang, Dakota T. Thompson, Trevor Williams, Vincent T. Wu, Kelsey E. Koch, Mikhail V. Kulak, Allison W. Lorenzen, and Edward H. Cho
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Transcriptome ,IκBα ,medicine.anatomical_structure ,Mammary gland ,Conditional gene knockout ,medicine ,Morphogenesis ,Stem cell ,Progenitor cell ,Biology ,RSPO1 ,Cell biology - Abstract
SUMMARYMammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2γ transcription factor, encoded byTfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Herein, we utilized an inducible, conditional knockout ofTfap2cto elucidate the role of AP-2γ in maintenance and differentiation of MaSCs. Loss of AP-2γ in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors.AP-2γ regulated the expression of genes known to be required for mammary development includingC/EBPβ, IκBα, andRspo1. As a result, AP-2γ-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2γ is required for maintenance of pluripotent MaSCs and their ability to develop mammary gland structures.HighlightsAP-2γ-deficient mice exhibited repressed ductal outgrowth and regenerative capacityLoss of AP-2γ reduced the number of mammary stem and luminal progenitor cellsAP-2γ target genes, includingC/EBPβ, IκBα, andRspo1, regulate mammary developmentAP-2γ is required for maintenance of pluripotent mammary stem cellseTOC blurbGu, Cho and colleagues utilized a conditional knockout ofTfap2cto examine transcriptional effects of AP-2γ on mammary stem cells. Single cell analysis demonstrated that AP-2γ-deficient mice have decreased numbers of mammary stem cells and alteration of genes required for mammary development includingC/EBPβ, IκBα, andRspo1. They demonstrate that AP-2γ is necessary for maintenance of pluripotent mammary stem cells.
- Published
- 2020
26. Whole-genome mutational burden analysis of three pluripotency induction methods
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Kristopher L. Nazor, Kunal Bhutani, Heng Dai, Andy Wing Chun Pang, Roy Williams, Mahendra S. Rao, Nicholas J. Schork, Jeanne F. Loring, Edward H. Cho, Ha Tran, Željko Džakula, and Han Cao
- Subjects
0301 basic medicine ,Cellular differentiation ,Science ,DNA Mutational Analysis ,Induced Pluripotent Stem Cells ,General Physics and Astronomy ,Genomics ,Biology ,medicine.disease_cause ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Gene mapping ,medicine ,Humans ,Induced pluripotent stem cell ,Genetics ,Mutation ,Multidisciplinary ,Cell Differentiation ,General Chemistry ,Fibroblasts ,3. Good health ,030104 developmental biology ,Reprogramming - Abstract
There is concern that the stresses of inducing pluripotency may lead to deleterious DNA mutations in induced pluripotent stem cell (iPSC) lines, which would compromise their use for cell therapies. Here we report comparative genomic analysis of nine isogenic iPSC lines generated using three reprogramming methods: integrating retroviral vectors, non-integrating Sendai virus and synthetic mRNAs. We used whole-genome sequencing and de novo genome mapping to identify single-nucleotide variants, insertions and deletions, and structural variants. Our results show a moderate number of variants in the iPSCs that were not evident in the parental fibroblasts, which may result from reprogramming. There were only small differences in the total numbers and types of variants among different reprogramming methods. Most importantly, a thorough genomic analysis showed that the variants were generally benign. We conclude that the process of reprogramming is unlikely to introduce variants that would make the cells inappropriate for therapy., It is feared that reprogramming may introduce DNA mutations. Here Bhutani et al. take three different reprogramming methods and using comparative whole genome analyses do identify nucleotide variations that are different in reprogrammed cells from the original fibroblasts, but none convey oncogenic potential.
- Published
- 2016
27. Local anesthetics inhibit kinesin motility and microtentacle protrusions in human epithelial and breast tumor cells
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Edward H. Cho, Michelle Peckham, Jennifer R. Yoon, Rebecca A. Whipple, Stuart S. Martin, Michael A. Matrone, and Eric M. Balzer
- Subjects
Cancer Research ,Pathology ,medicine.medical_specialty ,Tetracaine ,Green Fluorescent Proteins ,Kinesins ,Breast Neoplasms ,Video microscopy ,Vimentin ,macromolecular substances ,Biology ,Article ,Motor protein ,Cell Movement ,Tubulin ,Microtubule ,Cell Line, Tumor ,Cell Adhesion ,medicine ,Humans ,Anesthetics, Local ,Mammary Glands, Human ,Cells, Cultured ,Dose-Response Relationship, Drug ,Lidocaine ,Epithelial Cells ,Cell biology ,Oncology ,Invadopodia ,biology.protein ,Kinesin ,Female ,Filopodia ,medicine.drug - Abstract
Detached breast tumor cells produce dynamic microtubule protrusions that promote reattachment of cells and are termed tubulin microtentacles (McTNs) due to their mechanistic distinctions from actin-based filopodia/invadopodia and tubulin-based cilia. McTNs are enriched with vimentin and detyrosinated α-tubulin, (Glu-tubulin). Evidence suggests that vimentin and Glu-tubulin are cross-linked by kinesin motor proteins. Using known kinesin inhibitors, Lidocaine and Tetracaine, the roles of kinesins in McTN formation and function were tested. Live-cell McTN counts, adhesion assays, immunofluorescence, and video microscopy were performed to visualize inhibitor effects on McTNs. Viability and apoptosis assays were used to confirm the non-toxicity of the inhibitors. Treatments of human non-tumorigenic mammary epithelial and breast tumor cells with Lidocaine or Tetracaine caused rapid collapse of vimentin filaments. Live-cell video microscopy demonstrated that Tetracaine reduces motility of intracellular GFP-kinesin and causes centripetal collapse of McTNs. Treatment with Tetracaine inhibited the extension of McTNs and their ability to promote tumor cell aggregation and reattachment. Lidocaine showed similar effects but to a lesser degree. Our current data support a model in which the inhibition of kinesin motor proteins by Tetracaine leads to the reductions in McTNs, and provides a novel mechanism for the ability of this anesthetic to decrease metastatic progression.
- Published
- 2010
28. c-Src differentially regulates the functions of microtentacles and invadopodia
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Jana Slovic, Amanda E. Boggs, Michael A. Matrone, Rebecca A. Whipple, Eric M. Balzer, Stuart S. Martin, Susette C. Mueller, Toshiyuki Yoneda, Keyata Thompson, and Edward H. Cho
- Subjects
Cancer Research ,Breast Neoplasms ,Biology ,Microtubules ,Article ,3T3 cells ,CSK Tyrosine-Protein Kinase ,Extracellular matrix ,Mice ,chemistry.chemical_compound ,Microtubule ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Genetics ,medicine ,Animals ,Humans ,Cytoskeleton ,Molecular Biology ,Actin ,3T3 Cells ,Protein-Tyrosine Kinases ,Actins ,Extracellular Matrix ,Cell biology ,src-Family Kinases ,medicine.anatomical_structure ,SU6656 ,chemistry ,Invadopodia ,Cancer research ,Female ,Cell Surface Extensions ,Proto-oncogene tyrosine-protein kinase Src - Abstract
During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Observations of actin staining within thin, tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells prompted an investigation of whether McTNs are structural or functional analogs of invadopodia. We show here that MDA-MB-231 cells are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by expression of a constitutively-active c-Src kinase, and repressed by expression of dominant negative, catalytically-inactive form of c-Src. In contrast, expression of inactive c-Src significantly increased McTN formation. Direct inhibition of c-Src with the SU6656 inhibitor compound also significantly enhanced McTN formation, but suppressed invadopodia, including the appearance of F-actin cores and phospho-cortactin foci, as well as completely blocking focal degradation of extracellular matrix. Additionally, silencing of Tks5 in Src-transformed fibroblasts blocked invadopodia without affecting McTNs. Genetic modification of c-Src activity that promoted McTN formation augmented capillary retention of circulating tumor cells in vivo and rapid re-attachment of suspended cells in vitro, even though invadopodia were strongly suppressed. These results indicate that McTNs are capable of enhancing tumor cell reattachment, even in the absence of Tks5 and active Src, and define separate cytoskeletal mechanisms and functions for McTNs and invadopodia.
- Published
- 2010
29. Epithelial-to-Mesenchymal Transition Promotes Tubulin Detyrosination and Microtentacles that Enhance Endothelial Engagement
- Author
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Stuart S. Martin, Jennifer R. Yoon, Eric M. Balzer, Michael A. Matrone, Michele Vitolo, Jing Yang, Olga B. Ioffe, Rebecca A. Whipple, Kimberly C. Tuttle, and Edward H. Cho
- Subjects
Cancer Research ,Tubulin—tyrosine ligase ,Biopsy ,Breast Neoplasms ,macromolecular substances ,Biology ,Epithelium ,Article ,Mesoderm ,Twist transcription factor ,Circulating tumor cell ,Tubulin ,Detyrosination ,Cell Adhesion ,Humans ,Neoplasm Invasiveness ,Epithelial–mesenchymal transition ,Neoplasm Metastasis ,Cell adhesion ,Cytoskeleton ,Twist-Related Protein 1 ,Nuclear Proteins ,Immunohistochemistry ,Oncology ,Cancer research ,biology.protein ,Tyrosine ,Female - Abstract
Epithelial-to-mesenchymal transition (EMT) is associated with increased breast tumor metastasis; however, the specific mechanisms by which EMT promotes metastasis remain somewhat unclear. Despite the importance of cytoskeletal dynamics during both EMT and metastasis, very few current studies examine the cytoskeleton of detached and circulating tumor cells. Specific posttranslational α-tubulin modifications are critical for adherent cell motility and implicated in numerous pathologies, but also remain understudied in detached cells. We report here that EMT induced through ectopic expression of Twist or Snail promotes α-tubulin detyrosination and the formation of tubulin-based microtentacles in detached HMLEs. Mechanistically, EMT downregulates the tubulin tyrosine ligase enzyme, resulting in an accumulation of detyrosinated α-tubulin (Glu-tubulin), and increases microtentacles that penetrate endothelial layers to facilitate tumor cell reattachment. Confocal microscopy shows that microtentacles are capable of penetrating the junctions between endothelial cells. Suppression of endogenous Twist in metastatic human breast tumor cells is capable of reducing both tubulin detyrosination and microtentacles. Clinical breast tumor samples display high concordance between Glu-tubulin and Twist expression levels, emphasizing the coupling between EMT and tubulin detyrosination in vivo. Coordinated elevation of Twist and Glu-tubulin at invasive tumor fronts, particularly within ductal carcinoma in situ samples, establishes that EMT-induced tubulin detyrosination occurs at the earliest stages of tumor invasion. These data support a novel model where the EMT that occurs during tumor invasion downregulates tubulin tyrosine ligase, increasing α-tubulin detyrosination and promoting microtentacles that could enhance the reattachment of circulating tumor cells to the vascular endothelium during metastasis. Cancer Res; 70(20); 8127–37. ©2010 AACR.
- Published
- 2010
30. Antimitotic chemotherapeutics promote adhesive responses in detached and circulating tumor cells
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Rebecca A. Whipple, Eric M. Balzer, Edward H. Cho, Michael A. Matrone, and Stuart S. Martin
- Subjects
Cancer Research ,Paclitaxel ,Cell division ,Immunoblotting ,Fluorescent Antibody Technique ,Breast Neoplasms ,Antimitotic Agents ,Biology ,Microtubules ,Article ,Extracellular matrix ,chemistry.chemical_compound ,Circulating tumor cell ,Cell Line, Tumor ,Depsipeptides ,Cell Adhesion ,medicine ,Humans ,Cell adhesion ,Microscopy, Confocal ,Neoplastic Cells, Circulating ,medicine.disease ,Primary tumor ,Cell biology ,Oncology ,chemistry ,Cell culture ,Female ,Antimitotic Agent - Abstract
In the clinical treatment of breast cancer, antimitotic cytotoxic agents are one of the most commonly employed chemotherapies, owing largely to their antiproliferative effects on the growth and survival of adherent cells in studies that model primary tumor growth. Importantly, the manner in which these chemotherapeutics impact the metastatic process remains unclear. Furthermore, since dissemination of tumor cells through the systemic circulation and lymphatics necessitates periods of detached survival, it is equally important to consider how circulating tumor cells respond to such compounds. To address this question, we exposed both nontumorigenic and tumor-derived epithelial cell lines to two antitumor compounds, jasplakinolide and paclitaxel (Taxol), in a series of attached and detached states. We report here that jasplakinolide promoted the extension of microtubule-based projections and microtentacle protrusions in adherent and suspended cells, respectively. These protrusions were specifically enriched by upregulation of a stable post-translationally modified form of alpha-tubulin, and this occurred prior to, and independently of any reductions in cellular viability. Microtubule stabilization with Taxol significantly enhanced these effects. Additionally, Taxol promoted the attachment and spreading of suspended tumor cell populations on extracellular matrix. While the antiproliferative effects of these compounds are well recognized and clinically valuable, our findings that microfilament and microtubule binding chemotherapeutics rapidly increase the mechanisms that promote endothelial adhesion of circulating tumor cells warrant caution to avoid inadvertently enhancing metastatic potential, while targeting cell division.
- Published
- 2009
31. Vimentin Filaments Support Extension of Tubulin-Based Microtentacles in Detached Breast Tumor Cells
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Eric M. Balzer, Rebecca A. Whipple, Stuart S. Martin, Edward H. Cho, Jennifer R. Yoon, and Michael A. Matrone
- Subjects
Cancer Research ,Cell ,Breast Neoplasms ,Vimentin ,macromolecular substances ,Models, Biological ,Article ,Cytokeratin ,Tubulin ,Microtubule ,Cell Line, Tumor ,Cell Adhesion ,Tumor Cells, Cultured ,medicine ,Humans ,Cloning, Molecular ,Neoplasm Metastasis ,Cytoskeleton ,Intermediate filament ,biology ,Extracellular Matrix ,Cell biology ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,Oncology ,Tumor progression ,Mutation ,biology.protein ,Cancer research - Abstract
Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulin-based protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated α-tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated α-tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread. [Cancer Res 2008;68(14):5678–88]
- Published
- 2008
32. The heavy metal cadmium induces valosin-containing protein (VCP)-mediated aggresome formation
- Author
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Edward H. Cho, Nancy H. Colburn, Ji Ming Wang, Thomas P. Conrads, Zhen Xiao, Kunio Nagashima, Changcheng Song, Stephen J. Lockett, Ren-Ming Dai, Timothy D. Veenstra, Qing Wang, and Chou-Chi H. Li
- Subjects
Protein Folding ,Valosin-containing protein ,Cell Cycle Proteins ,Histone Deacetylase 6 ,Toxicology ,Article ,Histone Deacetylases ,Mass Spectrometry ,Inclusion bodies ,Protein structure ,Valosin Containing Protein ,Humans ,Cells, Cultured ,Adenosine Triphosphatases ,Inclusion Bodies ,Pharmacology ,biology ,Ubiquitin ,HDAC6 ,Protein Structure, Tertiary ,Cell biology ,Aggresome ,Proteasome ,biology.protein ,Histone deacetylase ,Cadmium ,Deacetylase activity - Abstract
Cadmium (Cd2+) is a heavy metal ion known to have a long biological half-life in humans. Accumulating evidence shows that exposure to Cd2+ is associated with neurodegenerative diseases characterized by the retention of ubiquitinated and misfolded proteins in the lesions. Here, we report that Cd2+ directly induces the formation of protein inclusion bodies in cells. The protein inclusion body is an aggresome, a major organelle for collecting ubiquitinated or misfolded proteins. Our results show that aggresomes are enriched in the detergent-insoluble fraction of Cd2+-treated cell lysates. Proteomic analysis identified 145 proteins in the aggresome-enriched fractions. One of the proteins is the highly conserved valosin-containing protein (VCP), which has been shown to colocalize with aggresomes and bind ubiquitinated proteins through its N domain (#1-200). Our subsequent examination of VCP's role in the formation of aggresomes induced by Cd2+ indicates that the C-terminal tail (#780-806) of VCP interacts with histone deacetylase HDAC6, a mediator for aggresome formation, suggesting that VCP participates in transporting ubiquitinated proteins to aggresomes. This function of VCP is impaired by inhibition of the deacetylase activity of HDAC6 or by over-expression of VCP mutants that do not bind ubiquitinated proteins or HDAC6. Our results indicate that Cd2+ induces the formation of protein inclusion bodies by promoting the accumulation of ubiquitinated proteins in aggresomes through VCP and HDAC6. Our delineation of the role of VCP in regulating cell responses to ubiquitinated proteins has important implications for understanding Cd2+ toxicity and associated diseases.
- Published
- 2008
33. Induction of the Formyl Peptide Receptor 2 in Microglia by IFN-γ and Synergy with CD40 Ligand
- Author
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Ji Ming Wang, Edward H. Cho, Keqiang Chen, Stephen J. Lockett, Pablo Iribarren, Lingzhi Zhang, Nancy M. Dunlop, Jian Huang, and Wanghua Gong
- Subjects
MAPK/ERK pathway ,CD40 Ligand ,Immunology ,Formyl peptide receptor 2 ,Interferon-gamma ,Mice ,Cell Movement ,medicine ,Animals ,Immunology and Allergy ,RNA, Messenger ,Receptor ,CD40 ,Formyl peptide receptor ,biology ,Microglia ,Tumor Necrosis Factor-alpha ,Drug Synergism ,Cell migration ,Receptors, Formyl Peptide ,Cell biology ,medicine.anatomical_structure ,Gene Expression Regulation ,biology.protein ,Signal transduction ,Signal Transduction - Abstract
Human formyl peptide receptor (FPR)-like 1 (FPRL1) and its mouse homologue mFPR2 are functional receptors for a variety of exogenous and host-derived chemotactic peptides, including amyloid beta 1-42 (Abeta(42)), a pathogenic factor in Alzheimer's disease. Because mFPR2 in microglial cells is regulated by proinflammatory stimulants including TLR agonists, in this study we investigated the capacity of IFN-gamma and the CD40 ligand (CD40L) to affect the expression and function of mFPR2. We found that IFN-gamma, when used alone, induced mFPR2 mRNA expression in a mouse microglial cell line and primary microglial cells in association with increased cell migration in response to mFPR2 agonists, including Abeta(42). IFN-gamma also increased the endocytosis of Abeta(42) by microglial cells via mFPR2. The effect of IFN-gamma on mFPR2 expression in microglial cells was dependent on activation of MAPK and IkappaB-alpha. IFN-gamma additionally increased the expression of CD40 by microglial cells and soluble CD40L significantly promoted cell responses to IFN-gamma during a 6-h incubation period by enhancing the activation of MAPK and IkappaB-alpha signaling pathways. We additionally found that the effect of IFN-gamma and its synergy with CD40L on mFPR2 expression in microglia was mediated in part by TNF-alpha. Our results suggest that IFN-gamma and CD40L, two host-derived factors with increased concentrations in inflammatory central nervous system diseases, may profoundly affect microglial cell responses in the pathogenic process in which mFPR2 agonist peptides are elevated.
- Published
- 2007
34. Calibration and standardization of the emission light path of confocal microscopes
- Author
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Edward H. Cho and Stephen J. Lockett
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Physics ,Photomultiplier ,Microscopy, Confocal ,Histology ,Microscope ,Laser scanning ,business.industry ,Dynamic range ,Photon counting ,Pathology and Forensic Medicine ,law.invention ,Optics ,Optical microscope ,Tubulin ,law ,Calibration ,Optoelectronics ,business ,Light-emitting diode - Abstract
Recently, there has been a large expansion in the usage of optical microscopes for obtaining quantitative information from biological samples in order to determine fundamental biological information such as molecular kinetics and interaction, and heterogeneity within cell populations. Consequently, we built a highly stable, uniform, isotropically emitting and convenient-to-use light source, and designed image analysis procedures for calibrating the emission light path of optical microscopes. We used the source and procedures to analyse the quantitative imaging properties of a widely used model of laser scanning confocal microscope. Results showed that the overall performance was as high as could be expected given the inherent limitations of the optical components and photomultiplier tubes. We observed that the photon detection efficiency did not vary with photomultiplier tube gain and that the highest dynamic range was achieved with relatively low gain and 12-bit digitization. Practical applications of the light source for checking the transmission of optical components in the emission light path are presented.
- Published
- 2006
35. Activation of Toll-like Receptor 2 on Microglia Promotes Cell Uptake of Alzheimer Disease-associated Amyloid β Peptide
- Author
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Pablo Iribarren, Jianhong Chen, Stephen J. Lockett, Keqiang Chen, Edward H. Cho, Ji Ming Wang, Nancy M. Dunlop, Wanghua Gong, and Jinyue Hu
- Subjects
Lipopolysaccharides ,Staphylococcus aureus ,MAP Kinase Signaling System ,Amyloid beta ,Blotting, Western ,Peptidoglycan ,Ligands ,Transfection ,Models, Biological ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Proinflammatory cytokine ,Mice ,Alzheimer Disease ,medicine ,Animals ,RNA, Messenger ,RNA, Small Interfering ,Receptors, Lipoxin ,Receptor ,Molecular Biology ,Inflammation ,Mitogen-Activated Protein Kinase 1 ,Toll-like receptor ,Amyloid beta-Peptides ,Microscopy, Confocal ,Mitogen-Activated Protein Kinase 3 ,Microglia ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Chemotaxis ,NF-kappa B ,Neurodegenerative Diseases ,Cell Biology ,Flow Cytometry ,Receptors, Formyl Peptide ,Molecular biology ,Toll-Like Receptor 2 ,TLR2 ,medicine.anatomical_structure ,Microscopy, Fluorescence ,biology.protein ,Calcium ,Signal transduction ,Peptides - Abstract
The human G-protein-coupled formyl peptide receptor-like 1 (FPRL1) and its mouse homologue mFPR2 mediate the chemotactic activity of a variety of polypeptides associated with inflammation and bacterial infection, including the 42-amino acid form of amyloid beta peptide (Abeta42), a pathogenic factor in Alzheimer disease. Because mFPR2 was inducible in mouse microglial cells by proinflammatory stimulants, such as bacterial lipopolysaccharide, a ligand for the Toll-like receptor 4 (TLR4), we investigated the role of TLR2 in the regulation of mFPR2. We found that a TLR2 agonist, peptidoglycan (PGN) derived from Gram-positive bacterium Staphylococcus aureus, induced considerable mFpr2 mRNA expression in a mouse microglial cell line and primary microglial cells. This was associated with a markedly increased chemotaxis of the cells in response to mFPR2 agonist peptides. In addition, activation of TLR2 markedly enhanced mFPR2-mediated uptake of Abeta42 by microglia. Studies of the mechanistic basis showed that PGN activates MAPK and IkappaBalpha, and the effect of PGN on induction of mFPR2 was dependent on signaling pathways via ERK1/2 and p38 MAPKs. The use of TLR2 on microglial cells by PGN was supported by the fact that N9 cells transfected with short interfering RNA targeting mouse TLR2 failed to show increased expression of functional mFPR2 after stimulation with PGN. Our results demonstrated a potentially important role for TLR2 in microglial cells of promoting cell responses to chemoattractants produced in lesions of inflammatory and neurodegenerative diseases in the brain.
- Published
- 2006
36. Epigenetic silencing of manganese superoxide dismutase (SOD-2) in KAS 6/1 human multiple myeloma cells increases cell proliferation
- Author
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Peter A. Clausen, Celine Pompeia, William L. Farrar, David R. Hodge, Edward H. Cho, Victor E. Marquez, Suneetha B. Thomas, and Benjamin Peng
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Cancer Research ,DNA repair ,DNA Methyltransferase Inhibitor ,Biology ,Gene Expression Regulation, Enzymologic ,Epigenesis, Genetic ,chemistry.chemical_compound ,Cell Line, Tumor ,Humans ,Gene silencing ,Gene Silencing ,Promoter Regions, Genetic ,Cell Proliferation ,Pharmacology ,chemistry.chemical_classification ,Regulation of gene expression ,Reactive oxygen species ,Superoxide Dismutase ,DNA Methylation ,Molecular biology ,Gene Expression Regulation, Neoplastic ,Oncology ,chemistry ,Zebularine ,DNA methylation ,Cancer cell ,Molecular Medicine ,Multiple Myeloma - Abstract
The generation of reactive oxygen species (ROS) by mitochondrial electron transport chain (ETC) and oxidative phosphorylation activity, has been linked to modifications of multiple molecular processes, including lipid peroxidation, signaling pathway and transcription factor modulation, and oxidative damage to DNA. Oxidative damage by endogenous ROS has been associated with the etiology of various pathological states. There are numerous reports that levels of manganese superoxide dismutase enzyme (MnSOD), an antioxidant enzyme responsible for the attenuation of ROS, are lowered in cancer cells, but the reasons for this reduction are poorly defined. Epigenetic silencing of genes involved in tumor suppression and DNA repair is known to occur in a variety of malignant cell types. Here we report that in the human multiple myeloma cell line KAS 6/1, the SOD-2 gene, encoding manganese superoxide dismutase, is epigenetically silenced as a result of promoter hypermethylation. The DNA methyltransferase inhibitor Zebularine reverses SOD-2 promoter methylation, increasing gene expression and enzyme levels. Infection of KAS 6/1 cells with a recombinant adenovirus carrying the MnSOD cDNA reduced the cell proliferation rate by approximately one-half, confirming the detrimental effects of epigenetic silencing of SOD-2 expression.
- Published
- 2005
37. Identification of Probabilistic Transcriptional Switches in the Ly49 Gene Cluster
- Author
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Gareth E. Davies, Deborah L. Hodge, Paul W. Wright, Stephen K. Anderson, Véronique Pascal, Mehrnoosh Abshari, Stephen J. Lockett, Ali Saleh, and Edward H. Cho
- Subjects
Genetics ,Regulation of gene expression ,Transgene ,Immunology ,Promoter ,Biology ,Cell biology ,Infectious Diseases ,Transcription (biology) ,Gene cluster ,Immunology and Allergy ,Coding region ,Gene ,Transcription factor - Abstract
Murine natural killer cells selectively express members of the Ly49 family of class I MHC receptors; however, the molecular mechanism controlling probabilistic expression of Ly49 proteins has not been defined. A pair of overlapping, divergent promoters discovered in the Ly49g gene functions as a molecular switch that can produce a forward transcript containing the coding region of the gene (on position) or a noncoding transcript in the opposite direction (off position), and this element maintains transcription in the chosen direction. Competition of C/EBP and TBP transcription factors for overlapping binding sites determines the relative strength of the competing promoters and the probability of transcription in a given direction. Similar elements precede all Ly49 family members, and the relative strength of the forward promoter in each inhibitory Ly49 gene correlates with the percentage of natural killer cells that express a given receptor, supporting a promoter competition model of selective gene activation.
- Published
- 2004
38. Automatic and Quantitative Measurement of Protein-Protein Colocalization in Live Cells
- Author
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Zachary C. Dobbin, Sylvain V. Costes, George N. Pavlakis, Edward H. Cho, Stephen J. Lockett, and Dirk Daelemans
- Subjects
Nucleolus ,Green Fluorescent Proteins ,Analytical chemistry ,Biophysics ,Receptors, Cytoplasmic and Nuclear ,Image processing ,Biology ,Karyopherins ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Exponential growth ,Spectroscopy, Imaging, Other Techniques ,Fluorescence Resonance Energy Transfer ,Image Processing, Computer-Assisted ,Humans ,030304 developmental biology ,0303 health sciences ,Pixel ,Colocalization ,Leptomycin ,Compartmentalization (psychology) ,Genes, rev ,Förster resonance energy transfer ,chemistry ,Fatty Acids, Unsaturated ,Biological system ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
We introduce a novel statistical approach that quantifies, for the first time, the amount of colocalization of two fluorescent-labeled proteins in an image automatically, removing the bias of visual interpretation. This is done by estimating simultaneously the maximum threshold of intensity for each color below which pixels do not show any statistical correlation. The sensitivity of the method was illustrated on simulated data by statistically confirming the existence of true colocalization in images with as little as 3% colocalization. This method was then tested on a large three-dimensional set of fixed cells cotransfected with CFP/YFP pairs of proteins that either co-compartmentalized, interacted, or were just randomly localized in the nucleolus. In this test, the algorithm successfully distinguished random color overlap from colocalization due to either co- compartmentalization or interaction, and results were verified by fluorescence resonance energy transfer. The accuracy and consistency of our algorithm was further illustrated by measuring, for the first time in live cells, the dissociation rate (kd) of the HIV-1 Rev/CRM1 export complex induced by the cytotoxin leptomycin B. Rev/CRM1 colocalization in nucleoli dropped exponentially after addition of leptomycin B at a rate of 1.25 3 10 � 3 s � 1 . More generally, this algorithm can be used to answer a variety of biological questions involving protein-protein interactions or co-compartmentalization and can be generalized to colocalization of more than two colors.
- Published
- 2004
- Full Text
- View/download PDF
39. Association of Lsh, a Regulator of DNA Methylation, with Pericentromeric Heterochromatin Is Dependent on Intact Heterochromatin
- Author
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Qingsheng Yan, Stephen J. Lockett, Kathrin Muegge, and Edward H. Cho
- Subjects
DNA (Cytosine-5-)-Methyltransferase 1 ,DNA Replication ,Euchromatin ,Heterochromatin ,Centromere ,Nuclear Localization Signals ,Transfection ,Cell Line ,Mice ,parasitic diseases ,Animals ,Constitutive heterochromatin ,DNA (Cytosine-5-)-Methyltransferases ,Heterochromatin organization ,Fluorescent Antibody Technique, Indirect ,Molecular Biology ,Sequence Deletion ,Transcriptional Regulation ,Mice, Knockout ,Genetics ,biology ,fungi ,EZH2 ,DNA Helicases ,Cell Biology ,DNA Methylation ,Recombinant Proteins ,Protein Structure, Tertiary ,Histone ,DNA methylation ,NIH 3T3 Cells ,biology.protein ,CpG Islands ,Heterochromatin protein 1 - Abstract
The eukaryotic genome is packaged into distinct domains of transcriptionally active euchromatin and silent heterochromatin. A hallmark of mammalian heterochromatin is CpG methylation. Lsh, a member of the SNF2 family, is a major regulator of DNA methylation in mice and thus crucial for normal heterochromatin formation. In order to define the molecular function of Lsh, we examined its cellular localization and its association with chromatin. Our studies demonstrate that Lsh is an exclusively nuclear protein, and we define a nuclear localization domain within the N-terminal portion of Lsh. Lsh strongly associates with chromatin and requires the internal and C-terminal regions for this interaction. Lsh accumulates at pericentromeric heterochromatin, suggesting a direct role for Lsh in the methylation of centromeric DNA sequences and the formation of heterochromatin. In search of a signal that is responsible for Lsh recruitment to pericentromeric heterochromatin, we found that histone tail modifications were critical. Prolonged treatment with histone deacetylase inhibitors has been reported to disrupt higher-order heterochromatin organization, and this was accompanied by dissociation of Lsh from pericentromeric heterochromatin. These results are consistent with a model in which Lsh is recruited by intact heterochromatin structure and then assists in maintaining heterochromatin organization by establishing CpG methylation patterns.
- Published
- 2003
40. CpG‐containing oligodeoxynucleotide promotes microglial cell uptake of amyloid β 1–42 peptide by up‐regulating the expression of the G‐protein‐coupled receptor mFPR2
- Author
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Badarch Uranchimeg, Stephen J. Lockett, Wanghua Gong, Pablo Iribarren, Keqiang Chen, Ji Ming Wang, Jinyue Hu, and Edward H. Cho
- Subjects
CpG Oligodeoxynucleotide ,Oligonucleotides ,Ligands ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Monocytes ,Receptors, G-Protein-Coupled ,Mice ,Genetics ,Animals ,Humans ,5-HT5A receptor ,Receptor ,Molecular Biology ,Protease-activated receptor 2 ,G protein-coupled receptor ,Inflammation ,Amyloid beta-Peptides ,Microscopy, Confocal ,Dose-Response Relationship, Drug ,Chemistry ,Chemotaxis ,TLR9 ,hemic and immune systems ,Receptors, Formyl Peptide ,Endocytosis ,Peptide Fragments ,Up-Regulation ,Cell biology ,Toll-Like Receptor 4 ,Gene Expression Regulation ,Pertussis Toxin ,Toll-Like Receptor 9 ,CpG Islands ,Microglia ,Peptides ,Relaxin/insulin-like family peptide receptor 2 ,Biotechnology - Abstract
Human G protein-coupled formyl peptide receptor like 1 (FPRL1) and its mouse homologue murine formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of amyloid beta 1-42 (Abeta42), a key pathogenic peptide in Alzheimer's disease (AD). Since mFPR2 is up-regulated in mouse microglia by lipopolysaccharide (LPS), a Toll-like receptor 4 ligand, we investigated the capacity of CpG-containing oligodeoxynucleotide (ODN), a Toll-like receptor (TLR) 9 ligand, to regulate the expression of mFPR2 in mouse microglia. CpG ODN markedly enhanced the expression and function of mFPR2 in microglial cells, which exhibited increased chemotactic responses to mFPR2 agonists, including Abeta42. The effect of CpG ODN is dependent on activation of p38 MAPK. Further studies showed that CpG ODN-treated microglia increased their capacity to endocytose Abeta42 through mFPR2, as this process was abrogated by pertussis toxin, a Gi protein inhibitor, and W peptide, another potent mFPR2 agonist. Our results suggest that TLR9 may play an important role in promoting microglial recognition of Abeta42, thus affecting the pathogenic process of AD.
- Published
- 2005
41. Circulating tumor cells as emerging tumor biomarkers in lung cancer
- Author
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Edward H, Cho
- Subjects
Circulating Tumor Cells - Published
- 2012
42. Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin
- Author
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Rebecca A. Whipple, Eric M. Balzer, Keyata Thompson, Amanda E. Boggs, Stuart S. Martin, Michael A. Matrone, Edward H. Cho, Jennifer R. Yoon, and Michele Vitolo
- Subjects
Cofilin 1 ,Cancer Research ,Class I Phosphatidylinositol 3-Kinases ,Mutation, Missense ,macromolecular substances ,Biology ,Article ,Gene Knockout Techniques ,Phosphatidylinositol 3-Kinases ,Microtubule ,Cell Line, Tumor ,Genetics ,Cell Adhesion ,Phosphoprotein Phosphatases ,PTEN ,Humans ,Cytoskeleton ,Cell adhesion ,Molecular Biology ,PI3K/AKT/mTOR pathway ,Actin ,PTEN Phosphohydrolase ,Lim Kinases ,Epithelial Cells ,Actomyosin ,Cofilin ,Actin cytoskeleton ,Cell biology ,biology.protein ,Cancer research ,Cell Surface Extensions ,Proto-Oncogene Proteins c-akt - Abstract
Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.
- Published
- 2012
43. Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR
- Author
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Vaughn V. Smider, Maria Loressa Uson, Peter Kuhn, Devin Sok, Stephanie A. Kazane, Peter G. Schultz, and Edward H. Cho
- Subjects
Receptor, ErbB-2 ,Oligonucleotides ,Biology ,Polymerase Chain Reaction ,Antibodies ,law.invention ,Nucleic acid thermodynamics ,chemistry.chemical_compound ,law ,Cell Line, Tumor ,Neoplasms ,Leukocytes ,Humans ,Polymerase chain reaction ,chemistry.chemical_classification ,Cell Nucleus ,Multidisciplinary ,Oligonucleotide ,Temperature ,Nucleic Acid Hybridization ,DNA ,Sequence Analysis, DNA ,Biological Sciences ,Molecular biology ,Amino acid ,Kinetics ,chemistry ,Biochemistry ,Microscopy, Fluorescence ,Cell culture ,Immune System ,biology.protein ,Antibody ,Conjugate - Abstract
Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2 + cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2 + cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.
- Published
- 2012
44. Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line
- Author
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Maria Loressa Uson, Melissa Torrey, Edward H. Cho, Peter Kuhn, Daniel C. Lazar, Mitchell E. Gross, Thomas J. Metzner, and Madelyn Luttgen
- Subjects
PCA3 ,Adult ,Male ,Indoles ,Biophysics ,Biology ,Article ,Cytokeratin ,Prostate cancer ,Circulating tumor cell ,Structural Biology ,Prostate ,Cell Line, Tumor ,LNCaP ,medicine ,Humans ,Fluorescent Antibody Technique, Indirect ,Molecular Biology ,Prostatic Neoplasms ,Cell Biology ,medicine.disease ,Neoplastic Cells, Circulating ,Androgen receptor ,medicine.anatomical_structure ,Cell culture ,Receptors, Androgen ,Immunology ,Cancer research ,Keratins ,Leukocyte Common Antigens - Abstract
Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate, and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity, and nuclear to cytoplasmic (N/C) ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.
- Published
- 2012
45. Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells
- Author
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Keyata Thompson, Stuart S. Martin, Michele Vitolo, Michael A. Matrone, Jennifer R. Yoon, Eric M. Balzer, Olga B. Ioffe, Rebecca A. Whipple, Edward H. Cho, Ming Tan, and Kimberly C. Tuttle
- Subjects
Cancer Research ,Microtubule-associated protein ,Tau protein ,Breast Neoplasms ,tau Proteins ,medicine.disease_cause ,Article ,Metastasis ,Circulating tumor cell ,Tubulin ,Cell Line, Tumor ,Genetics ,medicine ,Cell Adhesion ,Tumor Cells, Cultured ,Humans ,Neoplasm Invasiveness ,Neoplasm Metastasis ,Cytoskeleton ,Cell adhesion ,Molecular Biology ,biology ,Cancer ,medicine.disease ,Gene Knockdown Techniques ,Immunology ,biology.protein ,Cancer research ,Female ,Carcinogenesis - Abstract
The cytoskeletal organization of detached and circulating tumor cells is currently not well-defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, circulating tumor cells reattach in distant tissues via a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of circulating tumor cells match exactly with the mechanisms supporting tubulin microtentacles, which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how microtentacle formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to microtentacles and is both necessary and sufficient to promote microtentacle extension in detached breast tumor cells. Tau-induced microtentacles increase reattachment of suspended cells and retention of circulating tumor cells in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through microtentacle formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.
- Published
- 2010
46. Delocalization of γ-tubulin due to increased solubility in human breast cancer cell lines
- Author
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Eric M. Balzer, Rebecca A. Whipple, Edward H. Cho, Stuart S. Martin, and Michael A. Matrone
- Subjects
Cancer Research ,Breast Neoplasms ,macromolecular substances ,Microtubules ,Article ,Cell Line ,Cytosol ,Microtubule ,Tubulin ,Cell Line, Tumor ,Organelle ,Humans ,Fluorescent Antibody Technique, Indirect ,Microtubule nucleation ,Fluorescent Dyes ,Pharmacology ,Centrosome ,Organelles ,biology ,Tumor Cell Biology ,Cellular Structures ,Cell biology ,Oncology ,Solubility ,Cytoplasm ,biology.protein ,Molecular Medicine ,Benzimidazoles ,Female ,Cell fractionation ,Protein Processing, Post-Translational ,Subcellular Fractions - Abstract
The centrosome is the major organelle responsible for the nucleation and organization of microtubules into arrays. Recent studies demonstrate that microtubules can nucleate outside the centrosome. The molecular mechanisms controlling acentrosomal microtubule nucleation are currently poorly defined, and the function of this type of microtubule regulation in tumor cell biology is particularly unclear. Since microtubule nucleation is initiated by the gamma-tubulin protein, we examined the regulation of gamma-tubulin in a panel of human breast tumor cell lines, ranging from non-tumorigenic to highly aggressive. We have identified a more dispersive subcellular localization of gamma-tubulin in aggressive breast cancer cell lines, while gamma-tubulin localization remains largely centrosomal in non-invasive cell lines. Delocalization of gamma-tubulin occurs independently from changes in protein expression and is therefore regulated at the post-translational level. Subcellular fractionation revealed that tumor cell lines show an aberrantly increased release of gamma-tubulin into a soluble cytoplasmic fraction, with the most dramatic changes observed in tumor cell lines of greater metastatic potential. Extraction of soluble gamma-tubulin revealed acentrosomal incorporation of gamma-tubulin in cytoplasmic microtubules and along cell junctions. Moreover, acentrosomal delocalization of gamma-tubulin yielded resistance to colchicine-mediated microtubule collapse. These findings support a model where the solubility of gamma-tubulin can be altered through post-translational modification and provides a new mechanism for microtubule dysregulation in breast cancer. Gamma-tubulin which is delocalized from the centrosome can still clearly be incorporated into filaments, and defines a novel mechanism for tumor cells to develop resistance to microtubule-targeted chemotherapies.
- Published
- 2010
47. Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton
- Author
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Sylvain V. Costes, Chand Khanna, Ann F. Chambers, Jeffrey E. Green, Dalit Barkan, Hynda K. Kleinman, Mark J. Hoenorhoff, Justin L. Simmons, Anil K. Kamaraju, Holly Asmussen, Stephen J. Lockett, Edward H. Cho, and Zi-yao Liu
- Subjects
Cancer Research ,Myosin light-chain kinase ,Stress fiber ,Myosin Light Chains ,Fluorescent Antibody Technique ,macromolecular substances ,Biology ,Filamentous actin ,Article ,Mice ,Cell Line, Tumor ,Animals ,Neoplasm Metastasis ,Phosphorylation ,Cytoskeleton ,Myosin-Light-Chain Kinase ,Cell Proliferation ,DNA Primers ,Base Sequence ,Cell growth ,Cell Cycle ,Cell cycle ,Cell biology ,Fibronectins ,Enzyme Activation ,Oncology ,Cell culture ,Cancer research ,Cell Division ,Cancer dormancy - Abstract
Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin β1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin β1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241–50]
- Published
- 2008
48. Sphingomyelinase Restricts the Lateral Diffusion of CD4 and Inhibits Human Immunodeficiency Virus Fusion▿
- Author
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Danielle L. Guiffre, Stephen J. Lockett, Satinder S. Rawat, Catherine M. Finnegan, Alfred H. Merrill, Robert Blumenthal, and Edward H. Cho
- Subjects
Receptors, CXCR4 ,Receptors, CCR5 ,Anti-HIV Agents ,Immunology ,Virus Attachment ,Plasma protein binding ,Sphingomyelin phosphodiesterase ,Biology ,HIV Envelope Protein gp120 ,Endocytosis ,Microbiology ,CXCR4 ,Virus ,Diffusion ,Viral envelope ,Virology ,Humans ,Fluorescence recovery after photobleaching ,virus diseases ,Virus Internalization ,Cell biology ,Virus-Cell Interactions ,Sphingomyelin Phosphodiesterase ,Insect Science ,CD4 Antigens ,HIV-1 ,Sphingomyelin ,HeLa Cells ,Protein Binding - Abstract
Previously, we reported that treatment of cells with sphingomyelinase inhibits human immunodeficiency virus type 1 (HIV-1) entry. Here, we determined by measuring fluorescence recovery after photobleaching that the lateral diffusion of CD4 decreased 4-fold following sphingomyelinase treatment, while the effective diffusion rate of CCR5 remained unchanged. Notably, sphingomyelinase treatment of cells did not influence gp120 binding, HIV-1 attachment, or fluid-phase and receptor-mediated endocytosis. Furthermore, sphingomyelinase treatment did not affect the membrane disposition of the HIV receptor proteins CD4, CXCR4, and CCR5, as determined by Triton X-100 extraction. Restriction of CD4 diffusion by antibody cross-linking also inhibited HIV infection. We therefore interpret the decrease in CD4 lateral mobility following sphingomyelinase treatment in terms of clustering of CD4 molecules. Examination of fusion intermediates indicated that sphingomyelinase treatment inhibited HIV at a step in the fusion process after CD4 engagement. Maximal inhibition of fusion was observed following short coculture times and with target cells that express low levels of CD4. As HIV entry into cells requires the sequential engagement of viral envelope protein with CD4 and coreceptor, we propose that sphingomyelinase inhibits HIV infection by inducing CD4 clustering that prevents coreceptor engagement and HIV fusion.
- Published
- 2007
49. Secreted semaphorins modulate synaptic transmission in the adult hippocampus
- Author
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David D. Ginty, Jehuda P. Sepkuty, Richard L. Huganir, Alex L. Kolodkin, Edward H. Cho, Amar Sahay, and Chong Hyun Kim
- Subjects
animal structures ,Patch-Clamp Techniques ,Time Factors ,Neuropilins ,Blotting, Western ,Synaptophysin ,Nerve Tissue Proteins ,Semaphorins ,Biology ,Hippocampal formation ,In Vitro Techniques ,Hippocampus ,Synaptic Transmission ,Mice ,Prosencephalon ,Semaphorin ,Biological neural network ,Reaction Time ,Animals ,Humans ,Receptors, AMPA ,In Situ Hybridization ,Mice, Knockout ,Dose-Response Relationship, Drug ,General Neuroscience ,Dentate gyrus ,Age Factors ,Intracellular Signaling Peptides and Proteins ,Excitatory Postsynaptic Potentials ,Membrane Proteins ,Electroencephalography ,Neuropilin-1 ,Neuropilin-2 ,Rats ,Synaptic fatigue ,nervous system ,Animals, Newborn ,Synaptic plasticity ,embryonic structures ,sense organs ,Neural development ,Neuroscience ,Disks Large Homolog 4 Protein ,Subcellular Fractions ,Cellular/Molecular - Abstract
Modulation of synaptic activity is critical for neural circuit function and behavior. The semaphorins are a large, phylogenetically conserved protein family with important roles in neural development. However, semaphorin function in the adult brain has yet to be determined. Here, we show that the coreceptors for secreted semaphorins, the neuropilins, are found at synapses and localize to molecular layers of the adult mouse hippocampus and accessory olfactory cortex. Moreover, application of the secreted semaphorin Sema3F to acute hippocampal slices modulates both the frequency and amplitude of miniature EPSCs in granule cells of the dentate gyrus and pyramidal neurons of CA1. Finally, we show that mice lacking Sema3F are prone to seizures. These results suggest a novel role for semaphorins as synaptic modulators and illustrate the diverse repertoire of these guidance cues in both the formation and function of neural circuits.
- Published
- 2005
50. Osteoblast Adhesion on Tissue Engineering Scaffolds Made by Bio-Manufacturing Techniques
- Author
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J. J. Stone, Wei Sun, F. W. Wang, T. Dutta Roy, Steve Lockett, L. Henderson, and Edward H. Cho
- Subjects
Materials science ,Nanotechnology ,Osteoblast ,Cell migration ,Characterization (materials science) ,Focal adhesion ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Tissue engineering ,Polycaprolactone ,medicine ,Biomanufacturing ,Cell adhesion ,Biomedical engineering - Abstract
Scientific exploration into understanding and developing relationships between three-dimensional (3D) scaffolds prepared by rapid prototyping (RP) and cellular response has focused primarily on end results targeting osteoblast proliferation and differentiation. Here at the National Institute of Standards and Technology (NIST), we take a systems approach to developing relationships between material properties and quantitative biological responses. This study in particular focuses on the screening of parameters controlled by RP techniques and their ability to trigger signalling events leading to cell adhesion. This pioneering research in our group also characterizes the in vitro cell-material interactions of 2D films and 3D scaffolds. From there, one can postulate on contributory factors leading to cell migration, proliferation, and differentiation. In summary, we believe that the quantitative information from this fundamental investigation will enhance our knowledge of the interactions between cells and 3D material interfaces with respect to formation of focal adhesions. This work consists of two sections — the application of imaging techniques for 3D characterization of properties and culturing of osteoblasts for size and shape determination. This includes quantifying the number of focal adhesion sites. We are using 3D RP polycaprolactone (PCL) scaffolds as this surrogate model in which to compare 2D to 3D material performance and cell interactions. Using RP bio-manufacturing techniques to fabricate tissue engineering scaffolds allows for control of pore size, strut size, and layer thickness, therefore providing adjustable parameters to study which can potentially influence, or even dynamically modulate, cellular adhesion. Imaging results after culturing for 24 h showed differences in cell morphology and spreading relative to the different structures. The focal adhesion response also varied, indicating an apparent loss of organization in 3D scaffolds compared to 2D surfaces. See Results and Discussion for details.
- Published
- 2005
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