Transplantation of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors into Early-Stage Zebrafish Embryos.

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2 weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.

[Data protection in rehabilitation research]. / Datenschutz in der Rehabilitationsforschung.

Data protection is regulated by legislation and has to be adhered to by scientists, too. This overview shows where aspects of data protection have to be considered in rehabilitation research. Important legal sources are the code of social law X, the German Federal Data Protection Act and the data protection acts of the German states. Specific recommendations about patient information sheet and written informed consent are given for research based on interviews with study participants. Furthermore, operations such as collecting, processing, using, storing, publishing and archiving of personal data are explained, taking into account the requirements of data protection. A practical example (URL: www.thieme-connect.de/ejournals/toc/rehabilitation) shows how to separate personal data and research data using the services of an external data custodian.

Catch of the day: zebrafish as a human cancer model.

Zebrafish are making big waves in the field of cancer research. The effect has been widespread and continues to gain speed as more and more cancer researchers ride the wave of zebrafish biology. This has been largely due to the development of transgenic and xenograft models of cancer, which recapitulate many aspects of different human cancers including lymphoblastic T-cell leukemia, pancreatic cancer, melanoma and rhabdomyosarcoma. These models are already being utilized by academia and industry to search for genetic and chemical modifiers of cancer with success. The attention has been further stimulated by the amenability of zebrafish to pharmacological testing and the superior imaging properties of fish tissues that allow visualization of cancer progression and angiogenesis in live animals. This review summarizes the current zebrafish models of cancer and discusses their utility in human cancer research and future directions in the field.

Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.

The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.

Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk-CAS complexes.

c-Abl and the Abl-related gene product (Arg) are nonreceptor tyrosine kinases that regulate the actin cytoskeleton of cells by direct association with F-actin and localization to focal contacts. However, the biological significance of this interaction is not known. We show here that transfection of COS-7 cells with a kinase-inactive form of c-Abl (Abl) promotes c-Crk II/p130(CAS) (Crk-CAS) coupling, enhancing cell migration. Moreover, embryonic fibroblast cells isolated from mice devoid of endogenous Abl and Arg (abl-/- arg-/-) demonstrate increased Crk-CAS coupling and motility. Conversely, expression of a kinase-active form of Abl or reconstitution of abl-/- arg-/- cells with wild-type Abl prevents Crk-CAS coupling and inhibits cell migration. Thus, Abl and Arg kinases play a critical role in preventing cell migration through regulation of Crk and CAS adaptor protein complexes, which are necessary for cell movement.

Molecular signaling mechanisms of cell migration and invasion.

The ability of immune cells to migrate and invade the extracellular matrix (ECM) is a central process involved in immunologic surveillance as well as inflammation. We have shown that interaction of cells with adhesive proteins or growth factors (chemokines) present in the ECM control cell migration/invasion through activation of mitogen-activated protein kinases ERK1 and ERK2 and molecular coupling of the adapter proteins p130CAS and c-CrkII. During cell migration, ERK and CAS/Crk coupling operate as distinct signaling pathways that facilitate actin-myosin motor assembly and actin membrane ruffles, respectively. Furthermore, activation of these signaling pathways protects cells from apoptosis during invasion of the ECM, which is necessary as migratory cells colonize foreign sites in the body.

Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry.

The Crk-associated substrate, p130(CAS), has been implicated in the regulation of the actin cytoskeleton following ligation of cell integrins with the extracellular matrix. Integrin-mediated cell adhesion involves p130(CAS) association with focal adhesion kinase (p125(FAK)). Internalization/cell entry of type 2 and type 5 adenoviruses (Ad) is also mediated by alpha(v) integrins. However, expression of dominant negative forms of p125(FAK) does not alter virus entry, and Ad entry occurs normally in p125(FAK)-deficient fibroblasts. We now provide evidence that Ad internalization, a process which is mediated by alpha(v) integrins, also requires p130(CAS) and phosphatidylinositol-3-OH kinase (PI 3-kinase). Ad induces p130(CAS) phosphorylation and inhibition of p130(CAS) phosphorylation by tyrphostin and genistein, or expression of the substrate domain deleted p130(CAS) blocks Ad internalization. p130(CAS) was also found to associate with the p85 subunit of PI 3-kinase through its proline-rich domain during virus internalization and expression of p130(CAS) containing a deleted proline-rich domain (PRD) inhibited adenovirus cell entry. We showed further that the RPLPSPP motif in the proline-rich region of p130(CAS) interacts with the SH3 domain of p85/PI 3-kinase. These studies reveal the molecular basis by which p130(CAS) coordinates the signaling pathways involved in integrin-mediated Ad endocytosis.

Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix.

Regulation of cell migration/invasion is important for embryonic development, immune function, and angiogenesis. However, migratory cells must also coordinately activate survival mechanisms to invade the extracellular matrix and colonize foreign sites in the body. Although invasive cells activate protective programs to survive under diverse and sometimes hostile conditions, the molecular signals that regulate these processes are poorly understood. Evidence is provided that signals that induce cell invasion also promote cell survival by suppressing apoptosis of migratory cells. Extracellular-regulated kinase (ERK) activation and molecular coupling of the adaptor proteins p130 Crk-associated substrate (CAS) and c-CrkII (Crk) represent two distinct pathways that induce cell invasion and protect cells from apoptosis in a three-dimensional collagen matrix. CAS/Crk-mediated cell invasion and survival requires activation of the small GTPase Rac, whereas ERK-induced cell invasion, but not survival requires myosin light chain kinase activation and myosin light chain phosphorylation. Uncoupling CAS from Crk or inhibition of ERK activity prevents migration and induces apoptosis of invasive cells. These findings provide molecular evidence that during invasion of the extracellular matrix, cells coordinately regulate migration and survival mechanisms through ERK activation and CAS/Crk coupling.

ErbB2 is necessary for induction of carcinoma cell invasion by ErbB family receptor tyrosine kinases.

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.

Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase.

The small GTPase Rac is thought to regulate cell movement by influencing actin cytoskeletal organization and membrane ruffling. However, cell migration also depends on the activation of mitogen-activated protein kinase (MAPK), which can regulate myosin motor function, an event critical for cell contraction. Evidence is provided that, during active cell adhesion to the extracellular matrix, Rac potentiates the MAPK pathway and influences cell migration by selectively synergizing with Raf kinase but not with Ras or MAPK kinase. In fact, the synergy between Rac and Raf kinase increases the chemotactic sensitivity of cells to epidermal growth factor by 1000-fold. Therefore, the role of Rac in cell migration not only depends on its ability to regulate actin cytoskeletal organization but also on its capacity to potentiate chemokine activation of MAPK in a manner that depends on active cell adhesion to the extracellular matrix.

Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.

Four human ras homologs differ in their abilities to activate Raf-1, induce transformation, and stimulate cell motility.

Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras >/= Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A >/= N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998) J. Biol. Chem. 273, 1782-1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.

Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis.

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5-120 min) was refractory to integrin antagonists, whereas the sustained activity (4-20 h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin alphavbeta3.

CAS/Crk coupling serves as a "molecular switch" for induction of cell migration.

Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a "molecular switch" for the induction of cell migration and appears to contribute to the invasive property of tumors.

Adenovirus endocytosis via alpha(v) integrins requires phosphoinositide-3-OH kinase.

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by alpha(v) integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas alpha(v) integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with alpha(v), integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.

Regulation of cell motility by mitogen-activated protein kinase.

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.

Tumor cell interactions with adhesion proteins and growth factors likely contribute to the metastatic cascade. Evidence is provided that insulin or insulin-like growth factor-mediated signals cooperate with the commonly expressed integrin alpha v beta 5 to promote spontaneous pulmonary metastasis of multiple tumor cell types in both the chick embryo and severe combined immune deficiency mouse/human chimeric models. Expression of alpha v beta 5 in tumor cells promoted their adhesion to vitronectin in vitro. However, cell motility required cytokine stimulation, which caused redistribution of alpha-actinin to membrane-adhesive sites containing alpha v beta 5. Significantly, ligation of alpha v beta 5 and cytokine receptors were both required for spontaneous pulmonary metastasis of multiple tumor types even though it was not necessary for primary tumor growth. Thus, tumor cell metastasis can be regulated by a functional cooperation between cytokine signaling events and the adhesion receptor alpha v beta 5 in a manner independent of tumor cell growth. These findings provide evidence that integrin ligation, in conjunction with cytokine activation, plays an important role in the dissemination of malignant tumor cells.

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Receptor tyrosine kinase signaling required for integrin alpha v beta 5-directed cell motility but not adhesion on vitronectin.

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.

Comparison of the ontogeny of specific cell surface determinants on normal and delayed implanting mouse embryos.

The ontogeny of immunospecific cell surface determinants on preimplantation mouse embryos was determined by means of an antibody-dependent complement-mediated cell lysis assay. The determinants conferring sensitivity to lysis in that assay were first observed at the late blastocyst stage on embryos recovered from normal pregnancy on day 5 or grown to an equivalent stage in vitro. Although blastocysts recovered during the dormant phase associated with delayed implantation were not lysed, those recovered following reactivation with an injection of oestrogen to the mother were sensitive. Furthermore, it was found that treating the dormant embryos with neuraminidase rendered them sensitive to lysis. These results demonstrate that the appearance of specific cell surface determinants on mouse embryos is temporally associated with the process of attachment to the uterus in both normal pregnancy and at termination of the dormant phase associated with delayed implantation. They also indicate that those determinants may be 'masked' with sialic acid during embryonic diapause. It is suggested that such cell surface determinants could be important for embryo attachment and that the mechanism responsible for their expression may explain some aspects of the synchronization between the preimplantation conceptus and its mother at the time of implantation.