| 1. |
- Alkasalias, Twana, et al.
(author)
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RhoA knockout fibroblasts lose tumor-inhibitory capacity in vitro and promote tumor growth in vivo
- 2017
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:8, s. E1413-E1421
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Journal article (peer-reviewed)abstract
- Fibroblasts are a main player in the tumor-inhibitory microenvironment. Upon tumor initiation and progression, fibroblasts can lose their tumor-inhibitory capacity and promote tumor growth. The molecular mechanisms that underlie this switch have not been defined completely. Previously, we identified four proteins over-expressed in cancer-associated fibroblasts and linked to Rho GTPase signaling. Here, we show that knocking out the Ras homolog family member A (RhoA) gene in normal fibroblasts decreased their tumor-inhibitory capacity, as judged by neighbor suppression in vitro and accompanied by promotion of tumor growth in vivo. This also induced PC3 cancer cell motility and increased colony size in 2D cultures. RhoA knockout in fibroblasts induced vimentin intermediate filament reorganization, accompanied by reduced contractile force and increased stiffness of cells. There was also loss of wide F-actin stress fibers and large focal adhesions. In addition, we observed a significant loss of a-smooth muscle actin, which indicates a difference between RhoA knockout fibroblasts and classic cancer-associated fibroblasts. In 3D collagen matrix, RhoA knockout reduced fibroblast branching and meshwork formation and resulted in more compactly clustered tumor-cell colonies in coculture with PC3 cells, which might boost tumor stem-like properties. Coculturing RhoA knockout fibroblasts and PC3 cells induced expression of proinflammatory genes in both. Inflammatory mediators may induce tumor cell stemness. Network enrichment analysis of transcriptomic changes, however, revealed that the Rho signaling pathway per se was significantly triggered only after coculturing with tumor cells. Taken together, our findings in vivo and in vitro indicate that Rho signaling governs the inhibitory effects by fibroblasts on tumor-cell growth.
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| 2. |
- Bakhiet, M, et al.
(author)
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RANTES promotes growth and survival of human first-trimester forebrain astrocytes
- 2001
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In: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 3:2, s. 150-157
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Journal article (peer-reviewed)abstract
- We have examined the role of alpha and beta chemokines in the promotion of the ontogenetic development of the brain. RANTES was expressed preferentially in human fetal astrocytes in an age-dependent manner. Astrocytes from 5-week-old brains showed high proliferation and reduced survival, whereas 10-week-old astrocytes exhibited opposite effects. These effects were suppressed by anti-RANTES or anti-RANTES receptor antibodies and were enhanced by recombinant RANTES. RANTES induced tyrosine phosphorylation of several cellular proteins and nuclear translocation of STAT-1 in astrocytes. Interferons (IFN-gamma) was required for RANTES effects because RANTES induced IFN-gamma, and only 10-week-old astrocytes expressed the IFN-gamma receptor. Blocking of IFN-gamma with antibody reversed the effects of RANTES, indicating that cytokine/chemokine networks are critically involved in brain development.
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| 3. |
- Danielsson, Frida, et al.
(author)
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Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model
- 2013
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:17, s. 6853-6858
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Journal article (peer-reviewed)abstract
- The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.
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| 4. |
- Danielsson, Frida, et al.
(author)
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Vimentin Diversity in Health and Disease
- 2018
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In: Cells. - : MDPI. - 2073-4409. ; 7:10
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Research review (peer-reviewed)abstract
- Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medical aspects of intermediate filament proteins without particular commitment to vimentin, and other reviews have focussed on intermediate filaments in an in vitro context. In contrast, the present review focusses almost exclusively on vimentin, and covers both ex vivo and in vivo data from tissue culture and from living organisms, including a summary of the many phenotypes of vimentin knockout animals. Our aim is to provide a comprehensive overview of the current understanding of the many diverse aspects of vimentin, from biochemical, mechanical, cellular, systems biology and medical perspectives.
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| 5. |
- Evans, Caroline A., et al.
(author)
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Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome
- 2022
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:4
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Journal article (peer-reviewed)abstract
- Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI beta, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.
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| 6. |
- Fallqvist, Björn, 1985-, et al.
(author)
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Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts
- 2016
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In: Journal of The Mechanical Behavior of Biomedical Materials. - : Elsevier. - 1751-6161 .- 1878-0180. ; 59, s. 168-184
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Journal article (peer-reviewed)abstract
- In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively.
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| 7. |
- Gad, Annica
(author)
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Cell cycle control by components of cell anchorage
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle progression. In addition, we wanted to clarify if two components involved in cell anchorage, the hyaluronic acid (HA) -binding domain of CD44 and p21-activated kinase 1 (PAK1), can control cell cycle progression. Growth factors and cell anchorage have been considered to regulate cell proliferation exclusively by a joint control in the early/mid-G1-phase of the cell cycle. However, we found that in addition to this joint control, cell anchorage also controls progression through the late G1-phase as well as through the final cell division, cytokinesis. The control by cell anchorage in late G1-phase was found to be distinct from the control by growth factors in that it occurs after and independent of the normal control by serum, cyclin D-associated kinase activity, pRb and p107. In addition, we observed that although cells lacking anchorage could initiate the ingression of the cleavage furrow during cytokinesis, they could not complete cell division without cell anchorage. This anchoragedependent control of cytokinesis could be mediated by various integrins as well as by integrinindependent cell anchorage. Furthermore, we showed that the kinase-inhibitory domain of PAK1 could inhibit the induction of cyclin D1 and cyclin D2 as well as G1-phase progression. Surprisingly, the cell cycle inhibition of the PAK1 kinase-inhibitory domain appeared not to act through inhibiting PAK1 kinase activity, but through a different mechanism. We also found that the recombinant hyaluronic acid-binding domain of the cell surface receptor CD44 (CD44-HABD) could inhibit endothelial cell cycle progression, angiogenesis and tumour growth. A mutant CD44-HABD without the ability to disturb the binding of CD44 to hyaluronic acid also inhibited cell cycle progression. Therefore, we rule out the possibility that the observed cell cycle inhibition was due to loss of CD44 binding to hyaluronic acid. We hypothesize that the recombinant domain of CD44 inhibits cell cycle progression by binding to an unidentified cell surface ligand. A normal tissue cell can transform into a tumour cell only if it manages to overcome the control exerted by the surrounding extracellular matrix. Our results reveal that cell anchorage controls the cell cycle at additional steps as compared to growth factors. This finding underscores the importance of the cell anchorage-dependent control in the protection against cellular transformation and tumourigenesis. The anchorage of a cell to the surrounding extracellular matrix also needs to be altered at later stages of tumour progression. Thereby, further elucidation of how cell anchorage controls cell proliferation may provide means allowing the development of new therapies for cancer. This is further emphasised by our findings that domains of proteins involved in cell anchorage, such as CD44 and PAK1, can exert control of the cell cycle, and inhibit tumour cell growth.
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| 8. |
- Gad, Annica K. B., et al.
(author)
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Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions
- 2012
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In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:6, s. 2374-2382
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Journal article (peer-reviewed)abstract
- The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.
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| 9. |
- Gad, Annica K B, et al.
(author)
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RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules
- 2012
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In: Molecular Biology of the Cell. - Stockholm : Karolinska Institutet, Dept of Microbiology, Tumor and Cell Biology. - 1939-4586 .- 1059-1524.
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Journal article (peer-reviewed)abstract
- The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.
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| 10. |
- Nordgren, Niklas, et al.
(author)
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Oncogene induced stiffening of living cells
- 2015
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In: Abstracts of Papers of the American Chemical Society. - : AMER CHEMICAL SOC. - 0065-7727. ; 249
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Journal article (other academic/artistic)
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