Extensive characterization of sphere models established from colorectal cancer cell lines

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.     

Understanding microtubule dynamics for improved cancer therapy

Microtubules (MTs), key components of the cytoskeleton, are dynamic polymers of tubulin that form a well-organized network of polarized tube filaments. MT dynamics are highly regulated both spacially and temporally by several MT-related proteins, themselves regulated by several kinases and phosphatases via signaling cascades, and also by coordinated interactions with actin cytoskeleton and adhesion sites. Regulation of MT dynamics is crucial for mitosis, cell migration, cell signaling and trafficking. MT-targeted drugs (MTDs), which constitute a major anticancer drug family with antimitotic and antiangiogenic properties, inhibit tumor progression mainly by altering MT dynamics in both cancer and endothelial cells. Identification of proteins regulating the MT network will lead to a better understanding of tumor progression regulators and will be helpful in improving cancer therapy. Received 22 July 2005; received after revision 8 September 2005; accepted 12 September 2005     

不同转移潜能乳腺癌细胞中EGFR的表达及意义

目的 研究表皮生长因子受体（epidermal growth factor receptor,EGFR）在不同转移潜能乳腺癌细胞中的表达,并探讨其在乳腺癌侵袭转移过程中的作用. 方法 利用人工基质膜侵袭实验获得高、低转移潜能乳腺癌细胞亚系,四甲基偶氮唑盐（MTT）法检测两系细胞生长曲线和倍增时间,流式细胞仪检测两系细胞周期,transwell侵袭小室模型比较两系的迁移能力.应用逆转录聚合酶链反应（RT-PCR）和免疫印迹（Western Blot）检测EGFR在两系细胞中的表达. 结果 利用transwell小室成功筛选出高、低转移潜能乳腺癌细胞亚系;它们的体外生长速度、倍增时间、细胞周期和侵袭力具有明显差异;RT-PCR和Western Blot均显示在高转移潜能乳腺癌细胞中EGFR在基因和蛋白水平的表达均显著高于低转移乳腺癌细胞. 结论 EGFR的过表达与乳腺癌细胞侵袭能力显著性相关,EGFR在乳腺癌侵袭过程中发挥了重要的作用.     

Myosin II in mechanotransduction: master and commander of cell migration, morphogenesis, and cancer

Mechanotransduction encompasses the role of mechanical forces in controlling cell behavior by activating signal transduction pathways. Most forces at a cellular level are caused by myosin II, which contracts and cross-links actin. Myosin II-dependent forces are transmitted through the actin cytoskeleton to molecular endpoints that promote specific cellular outcomes, e.g., cell proliferation, adhesion, or migration. For example, most adhesive and migratory phenomena are mechanically linked by a molecular clutch comprised of mechanosensitive scaffolds. Myosin II activation and mechanosensitive molecular mechanisms are finely tuned and spatiotemporally integrated to coordinate morphogenetic events during development. Mechanical events dependent on myosin II also participate in tumor cell proliferation, invasion, and metastatic dissemination. Specifically, tumor cells alter the mechanical properties of the microenvironment to create favorable conditions for proliferation and/or dissemination. These observations position myosin II-dependent force generation and mechanotransduction at the crossroads between normal development and cancer.     

Filamentous actin accumulates during plant cell penetration and cell wall plug formation in <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration.     

Multiple roles for the actin cytoskeleton during regulated exocytosis

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e., secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane, and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules.     

Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

Pseudomonas aeruginosa is a major human opportunistic pathogen and one of the most important causal agents of bacteremia. For non-blood-borne infection, bacterial dissemination requires the crossing of the vascular endothelium, the main barrier between blood and the surrounding tissues. Here, we investigated the effects of P. aeruginosa type 3 secretion effectors, namely ExoS, ExoT, and ExoY, on regulators of actin cytoskeleton dynamics in primary endothelial cells. ExoS and ExoT similarly affected the Lim kinase-cofilin pathway, thereby promoting actin filament severing. Cofilin activation was also observed in a mouse model of P. aeruginosa-induced acute pneumonia. Rho, Rac, and Cdc42 GTPases were sequentially inactivated, leading to inhibition of membrane ruffling, filopodia, and stress fiber collapse, and focal adhesion disruption. At the end of the process, ExoS and ExoT produced a dramatic retraction in all primary endothelial cell types tested and thus a rupture of the endothelial monolayer. ExoY alone had no effect in this context. Cell retraction could be counteracted by overexpression of actin cytoskeleton regulators. In addition, our data suggest that moesin is neither a direct exotoxin target nor an important player in this process. We conclude that any action leading to inhibition of actin filament breakdown will improve the barrier function of the endothelium during P. aeruginosa infection.     

Tetraspanin-enriched microdomains regulate digitation junctions

Tetraspanins co-emerged with multi-cellular organisms during evolution are typically localized at the cell–cell interface, and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell–cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factor receptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell–matrix adhesion and cell–cell adhesion and involves both cell–cell and cell–matrix adhesion molecules.     

Actin dynamics in the regulation of endothelial barrier functions and neutrophil recruitment during endotoxemia and sepsis

Sepsis is a leading cause of death worldwide. Increased vascular permeability is a major hallmark of sepsis. Dynamic alterations in actin fiber formation play an important role in the regulation of endothelial barrier functions and thus vascular permeability. Endothelial integrity requires a delicate balance between the formation of cortical actin filaments that maintain endothelial cell contact stability and the formation of actin stress fibers that generate pulling forces, and thus compromise endothelial cell contact stability. Current research has revealed multiple molecular pathways that regulate actin dynamics and endothelial barrier dysfunction during sepsis. These include intracellular signaling proteins of the small GTPases family (e.g., Rap1, RhoA and Rac1) as well as the molecules that are directly acting on the actomyosin cytoskeleton such as myosin light chain kinase and Rho kinases. Another hallmark of sepsis is an excessive recruitment of neutrophils that also involves changes in the actin cytoskeleton in both endothelial cells and neutrophils. This review focuses on the available evidence about molecules that control actin dynamics and regulate endothelial barrier functions and neutrophil recruitment. We also discuss treatment strategies using pharmaceutical enzyme inhibitors to target excessive vascular permeability and leukocyte recruitment in septic patients.     

MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.     

Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on cancer cells

Protein farnesylation, catalyzed by protein farnesyltransferase, plays important roles in the membrane association and protein-protein interaction of a number of eukaryotic proteins. Recent development of farnesyltransferase inhibitors (FTIs) has led to further insight into the biological significance of farnesylation in cancer cells. A number of reports point to the dramatic effects FTIs exert on cancer cells. In addition to inhibiting anchorage-independent growth, FTIs cause changes in the cell cycle either at the G1/S or at the G2/M phase. Furthermore, induction of apoptosis by FTIs has been reported. FTIs also affects the actin cytoskeleton and cell morphology. This review summarizes these reports and discusses implications for farnesylated proteins responsible for these FTI effects. Received 17 April 2001; received after revision 28 May 2001; accepted 28 May 2001     

Transient cytoskeletal alterations after SOD1 depletion in neuroblastoma cells

We have studied the effects of superoxide production after Cu,Zn superoxide dismutase (SOD1) down-regulation by RNA interference. We demonstrated that SOD1 depletion induced, only in neuroblastoma cells, a decrease in actin and β-tubulin content and accumulation of neurofilament light chain and Tau proteins. Alterations of cell morphology and the microfilament network were also observed, together with the up-regulation of the Cdk5/p35 pathway, which is involved in the regulation of actin polymerization. The decrease of filamentous actin was transient and was recovered through the activation of p38/Hsp27 MAPK pathway, as well as after treatment with N-acetyl-L-cysteine. The importance of p38 in the recovery of cytoskeleton was confirmed by experiments carried out in the presence of its inhibitor SB203580, which induced cell death. Our data demonstrate that SOD1 is essential for the preservation of cytoskeleton integrity, by maintaining physiological concentration of reactive oxygen species and inhibiting the activation of the neuronal specific Cdk5/p35 pathway. P. Vigilanza, K. Aquilano: Both authors equally contributed to this work. Received 15 November 2007; received after revision 19 January 2008; accepted 22 January 2008     

The glycerophosphoinositols: cellular metabolism and biological functions

The glycerophosphoinositols are cellular products of phospholipase A₂ and lysolipase activities on the membrane phosphoinositides. Their intracellular concentrations can vary upon oncogenic transformation, cell differentiation and hormonal stimulation. Specific glycerophosphodiester phosphodiesterases are involved in their catabolism, which, as with their formation, is under hormonal regulation. With their mechanisms of action including modulation of adenylyl cyclase, intracellular calcium levels, and Rho-GTPases, the glycerophosphoinositols have diverse effects in multiple cell types: induction of cell proliferation in thyroid cells; modulation of actin cytoskeleton organisation in fibroblasts; and reduction of the invasive potential of tumour cell lines. More recent investigations include their effects in inflammatory and immune responses. Indeed, the glycerophosphoinositols enhance cytokine-dependent chemotaxis in T-lymphocytes induced by SDF-1α-receptor activation, indicating roles for these compounds as modulators of T-cell signalling and T-cell responses.     

Cytoskeleton–membrane interactions in membrane raft structure

Cell membranes are structurally heterogeneous, composed of discrete domains with unique physical and biological properties. Membrane domains can form through a number of mechanisms involving lipid–lipid and protein–lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft. How rafts form remains a current topic in membrane biology. We review here evidence of structuring of rafts by the cortical actin cytoskeleton. This includes evidence that the actin cytoskeleton associates with rafts, and that many of the structural and functional properties of rafts require an intact actin cytoskeleton. We discuss the mechanisms of the actin-dependent raft organization, and the properties of the actin cytoskeleton in regulating raft-associated signaling events. We end with a discussion of membrane rafts and the actin cytoskeleton in T cell activation, which function synergistically to initiate the adaptive immune response.     

Emodin inhibits tumor cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway

Enhanced cell migration is one of the underlying mechanisms in cancer invasion and metastasis. Therefore, inhibition of cell migration is considered to be an effective strategy for prevention of cancer metastasis. We found that emodin (3-methyl-1,6,8-trihydroxyanthraquinone), an active component from the rhizome of Rheum palmatum, significantly inhibited epidermal growth factor (EGF)- induced migration in various human cancer cell lines. In the search for the underlying molecular mechanisms, we demonstrated that phosphatidylinositol 3-kinase (PI3K) serves as the molecular target for emodin. In addition, emodin markedly suppressed EGF-induced activation of Cdc42 and Rac1 and the corresponding cytoskeleton changes. Moreover, emodin, but not LY294002, was able to block cell migration in cells transfected with constitutively active (CA)-Cdc42 and CA-Rac1 by interference with the formation of Cdc42/Rac1 and the p21-activated kinase complex. Taken together, data from this study suggest that emodin inhibits human cancer cell migration by suppressing the PI3K-Cdc42/Rac1 signaling pathway.Received 7 February 2005; received after revision 11 March 2005; accepted 18 March 2005     

The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases

Proteins are typically categorized into protein families based on their domain organization. Yet, evolutionarily unrelated proteins can also be grouped together according to their common functional roles. Sequestering proteins constitute one such functional class, acting as macromolecular buffers and serving as an intracellular reservoir ready to release large quantities of bound proteins or other molecules upon appropriate stimulation. Another functional protein class comprises effector proteins, which constitute essential components of many intracellular signal transduction pathways. For instance, effectors of small GTP-hydrolases are activated upon binding a GTP-bound GTPase and thereupon participate in downstream interactions. Here we describe a member of the IQGAP family of scaffolding proteins, DGAP1 from Dictyostelium, which unifies the roles of an effector and a sequestrator in regard to the small GTPase Rac1. Unlike classical effectors, which bind their activators transiently leading to short-lived signaling complexes, interaction between DGAP1 and Rac1-GTP is stable and induces formation of a complex with actin-bundling proteins cortexillins at the back end of the cell. An oppositely localized Rac1 effector, the Scar/WAVE complex, promotes actin polymerization at the cell front. Competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. We discuss the consequences of the dual roles exerted by DGAP1 and Rac1 in the regulation of cell motility and polarity, and propose that similar signaling mechanisms may be of general importance in regulating spatiotemporal dynamics of the actin cytoskeleton by small GTPases.     

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27^Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.     

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. Received 28 May 2008; received after revision 8 July 2008; accepted 29 July 2008     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006