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-Time-lapse videomicroscopy. 50 µl of a neutrophil granulocyte or tumor cell suspension was mixed with 100 µl of a buffered collagen solution and was subsequently filled into self-constructed migration chambers. After polymerization the chamber was sealed and placed under a second chamber which was constantly heated to 37 °C. The locomotory behaviour of the cells was recorded by a timelapse videorecorder connected to a videocamera.
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The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which functi...
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... videomicroscopy and computer-assisted cell tracking Collagen lattices were generated as previously described 4 . Briefly, 200,000 neutrophil granulocytes or 60,000 tumor cells were mixed with 100 µl of buffered collagen solution (pH 7.4) containing 1.67 mg/ml bovine collagen type I (Invitrogen, Cohesion Technologies, Palo Alto, CA) in minimal essential Eagle's medium (Flow, McLean, VA). The suspension was filled into self-constructed chambers 5 so that the chambers were filled to the half, and were allowed to polymerize for 20 min at 37 °C and 5% CO 2 humidified atmosphere ( Fig. 2). After polymerization interleukin-8 (Pharma Biotechnologie Hannover, Germany) was filled into the second half of the chamber at the double of the desired final concentration (2 to 2.000 ng/ml) for the experiments with neutrophil granulocytes and 100 ng/ml epidermal growth factor (EGF; Calbiochem, Bad Soden, German) for the experiments with tumor cells. For control experiments, the chambers were filled with medium alone. granulocyte or tumor cell suspension was mixed with 100 µl of a buffered collagen solution and was subsequently filled into self-constructed migration chambers. After poly- merization the chamber was sealed and placed under a second chamber which was constantly heated to 37 °C. The locomotory behaviour of the cells was recorded by a time- lapse videorecorder connected to a ...
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... behaviour of the cells within the three-dimensional collagen matrix was recorded at a 80 fold time-lapse mode for neutrophil granulocytes or a 1920 fold time-lapse mode for tumor cells, due to the differences in the migration velocity. The temperature of the chambers containing the collagen lattices was continuously kept at 37 °C (Fig. 2). After recording, thirty cells were randomly selected and the paths were digitized by computer-assisted cell tracking. From the digitized paths, the migratory activity of the population was calculated: the migratory activity is represented by the time-average of that part of the observed population (in percent), which was locomotory active at each given time point (one minute intervals for neutrophil granulocytes and 20 minute intervals for tumor cells). In contrast, the displacement (translocating cells in percent) represents, which part of the population was locomotory active within the whole observation period, thus, whether a cell moved or not, regardless how high the locomotory activity of each single-cell was. The locomotory activity of each single cell is at least represented by the time locomoting (in percent): the time locomoting is that part of the observation period, that the cell was actually locomotory active excluding pauses. In addition, the number of cell divisions that were observed within the tumor cell population was counted and calculated as cell divisions in percent of EGF-treated cells compared to untreated control ...
Citations
... The actin cytoskeleton is one of the central mechanical components responsible for the motility of cells, and its' analysis is an effective method of determining a migratory phenotype. Actin polymerization in migration is induced by the PI3-kinase and the PLC-γ / PKC cascade in a variety of cell types [119,125126127, including HSCs. So far several groups have convincingly demonstrated that SDF-1α induces actin polymerization [5, 39] as well as tyrosine phosphorylation of several components of focal adhesion complexes such as paxillin, the related adhesion focal tyrosine kinase (RAFTK/ pyk2), p130 cas , Crk-II, and Crk-L [122, 123]. ...
Migration is an innate and fundamental cellular function that enables hematopoietic stem cells (HSCs) and endothelial progenitors (EPCs) to leave the bone marrow, relocate to distant tissue, and to return to the bone marrow. An increasing number of studies demonstrate the widening scope of the therapeutic potential of both HSCs and endothelial cells. Therapeutic success however not only relies upon their ability to repair damaged tissue, but is also fundamentally dependent on the migration to these areas. Extensive in vivo and in vitro research efforts have shown that the most significant effects seen on HSC migration are initiated by the chemokine SDF-1alpha. In this review we will elucidate the many cellular and systemic factors of HSC and EPC cell migration and their modi operandi.
... With regard to our results, this view has to be rediscussed, because our results proved a promigratory effect of formyl peptides and IL-8 in correspondence with an increase of cAMP. We argue that the function of IL-8 as a start or stop-signal depends on the concentration of cAMP regulating the cytosolic calcium concentration [32]. Accordingly, Elferink and De Koster [33] recently termed cAMP as a modulator for calcium-dependent neutrophil migration. ...
... Thus, fMLP-mediated activation of G proteins causes two opposite effects on the regulation of the cellular calcium (Fig. 5): a PLCb2-mediated increase of IP 3 leads to a release of calcium from intracellular stores [34], whereas an adenylyl cyclase-dependent activation of the calcium pump SERCA via cAMP, PKA and phospholamban sequestrates cytosolic calcium. We conclude that the differential start-/stop-signalling arises from the bifurcation of the chemokine signalling, which we previously suggested as a 'two wheel model' for the regulation of cytosolic calcium (Fig. 5) [32]; the release of calcium from intracellular stores is ATP-independent. The second Figure 3 Changes of cellular cyclic adenosine monophosphate (cAMP) upon treatment of neutrophils with cholera toxin, pertussis toxin, formylmethionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). ...
Migration is a key function of stem cells during ontogenesis, of fibroblasts in wound healing and of immune cells in host defence. The signals that initiate migration are as important as signals that terminate migration, once the destination has been reached. We now show that formyl-methionyl-leucyl-phenylalanine (fMLP)-induced migration of neutrophils was inhibited by increasing concentrations of interleukin-8 (IL-8). IL-8 dose dependently increased the frequency and the duration of stop-periods, whereas the percentage of cells of a population that was locomotory active remained constant. The stop-signal delivered by IL-8 was intracellularly transduced by a dichotomic pathway: (i) the activation of the adenylyl cyclase leads to an increase of cytosolic cyclic adenosine monophosphate, which results in an activation of the sarcoplasmatic/endoplasmatic reticulum calcium ATPase pump and a calcium sequestration; (ii) the activation of the phospholipase Cbeta (PLCbeta) generates inositol-1,4,5-phosphate (IP3) and diacylglycerol (DAG), which results in IP3-mediated release of intracellularly stored calcium in the endoplasmatic reticulum and DAG-mediated activation of protein kinase C. Thus, we show for the first time that a chemokine, IL-8, in concert with fMLP, downregulates the neutrophil migration through the regulation of the intracellular calcium concentration via the adenylyl cyclase and the PLCbeta2.
... We previously elucidated the migratory activity of several human colon carcinoma cell lines 13 and breast carcinoma cell lines, 14 and compared the migratory activity of these cells with the T24 bladder carcinoma cells; 15 the T24 cells revealed the highest locomotor activity. Herein, we investigated the regulatory signal transduction for migration in T24 human bladder carcinoma cells to understand the underlying mechanisms of locomotion. ...
T24 human bladder carcinoma cells reveal a high locomotor activity (70% locomoting cells) within a 3-dimensional collagen matrix. This high migratory activity is induced by an autocrine engagement of the interleukin-8 receptor A, as was shown by antibodies neutralizing the secreted interleukin-8. Treatment of the cells with these specific antibodies reduced the locomotor activity by half. The intracellular signal transduction underlying the interleukin-8-induced T24 locomotion involves the activity of protein tyrosine kinases (PTKs), the phospholipase Cgamma (PLCgamma) and the protein kinase C (PKC), as proven by the use of specific enzyme inhibitors. These results suggest the following model for the regulatory signal transduction of interleukin-8-induced human T24 bladder carcinoma cell migration: The engagement of the interleukin-8-receptor, a receptor of the serpentine family, leads to the beta-arrestin-mediated activation of PTKs. These kinases phosphorylate the PLCgamma, which generates the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP(3)). DAG activates the PKC, whereas IP(3) mediates the release of calcium from the endoplasmatic reticulum. By means of confocal laser microscopy, we observed an oscillation of the cytosolic calcium concentration in migrating T24 cells, which were loaded with the calcium-dye fluo-3/AM. Here, we report on a new autocrine function of secreted interleukin-8 and the intracellular signal transduction leading to the regulation of cytosolic calcium and to a migratory tumor cell phenotype.
Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2⁻) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2⁻ release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2⁻. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs’ function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell.
The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.
Mistletoe therapy has been implemented in cancer therapy in Germany for the last 30 years, whereby its application in the clinic ranges between 46 to 70% of patients with malignant diseases including follicular B Non-Hodgkin's Lymphoma (B-NHL) and breast cancer. In the majority of cases, mistletoe extracts (also named Viscum album (VA) extracts) are given as an adjuvant therapy, e.g. in combination with chemotherapy. The use of VA extracts in the treatment of follicular B-NHL is still subject to controversial discussions. On the one hand, various studies indicated that intravenous application caused elevated interleukin-6 (IL-6) serum levels in patients, which would be fatal for B-NHL patients since IL-6 is a proliferation factor for neoplastic B-cells. On the other hand, we and other groups have shown that the VA extract Iscador®P does not provoke an IL-6 deregulation in follicular B-NHL cell lines and that subcutaneous application of Iscador®P does not cause increased IL-6 serum levels. Here we investigated the influence of the VA extract Iscador®P on the IL-6 induced proliferation of two follicular B-NHL cell lines. Interestingly, Iscador®P had a more profound inhibitory effect on the proliferation of follicular B-NHL cell lines if these were stimulated with IL-6. Thereby, Iscador®P acts differently on the investigated cell lines. For instance, in the WSU-NHL B-NHL cell line, Iscador®P and IL-6 co-treatment caused a bax up-regulation, which correlated with an increased number of apoptotic cells. In contrast, an increased number of apoptotic cells was not detectable in Sc-1 cells, albeit the proliferation rate of Iscador®P and IL-6 co-treated cells was markedly decreased. The observation that VA extracts are more potent if applied with proliferatory stimulus was also observed for the human breast cancer cell line MDA-MB-468-HER2. Here, the VA extract Iscador®M efficiently counteracts the EGF induced proliferation and migration of these cells. In summary, our data provide new insights in the potency of VA extracts for cancer treatment.
Within the past years, our knowledge about stem cells in health and disease has changed dramatically. To date, it is feasible to isolate and propagate human pluripotent stem cells from various sources, such as cord blood, bone marrow or adipose tissue, and to generate donor-specific ethically harmless induced pluripotent stem cells, which exhibits embryonic stem cell properties. However, irrespective of the used stem cell type(s), the success of tissue regeneration therapies does not only depend on the cells’ differentiation capacity, but also on their ability to migrate. Without migration, stem cells would neither be able to reach the appropriate degenerated tissue (if administered intravenously) nor they would be able to regenerate it because restoration of organ tissue integrity and function means to reconstruct a three-dimensional organ environment. However, the ability of stem cells to migrate is not only crucial for tissue regeneration processes, but do also play a role in tumor progression. To date, we know that cancer has its origin in a small subpopulation of cancer cells exhibiting stem cell properties, the so-called cancer stem cells. Because of their tumor initiation capacity, cancer stem cells have now also been linked to metastasis formation, which prerequisites cell migration. In summary, stem cell migration is a crucial process in both health and disease.Copyright © 2010 S. Karger AG, Basel
This chapter discusses the various methods used in the circulating cancer cells— namely, flow cytometry, video microscopy, and confocal laser scanning microscopy. Cancer is the second most prevalent cause of death and the formation of metastasis is the primary cause of death in cancer. Metastasizing tumor cells must traverse natural barriers, such as connective tissue components and organ epithelia at multiple stages of the metastatic process. The events, leading to metastasis are also discussed in the chapter. The process of extravasation, the active migration of tumor cells across the endothelial barrier, is described as a rate-regulating event in metastasis. The sequential steps of tumor cell extravasation are mainly based on in vitro models, using fixed sample preparations and electron microscopy. An in vitro model to study the extravasation of tumor cell clusters is described in the chapter. The advantage of this in vitro model is that the transmigration process is viewed perpendicular to the endothelial monolayer that allows the visualization of endothelial cell-tumor cell interactions, with the aid of fluorescence staining and live microscopy.
The ability to migrate is an innate and fundamental function of hematopoietic stem/progenitor cells (HSPCs) enabling them to leave and to return to the bone marrow, as well as to be recruited to injured tissues. The latter property of HSPCs concomitantly with their ability to transdifferentiate, thereby restoring the integrity of damaged tissues, raised great expectations for regenerative medicine purposes. It is well recognized that the migration of HSPCs is initiated and maintained by the chemokine stromal cell-derived factor-1α (SDF-1α). SDF-1α is expressed by bone marrow stroma cells, thereby generating a gradient, which directs the HSPC homing to the bone marrow. Likewise, SDF-1α is released by endothelial cells in close proximity of damaged organ tissue, thereby attracting HSPCs to sites of injury, which thereupon participate in tissue repair. It is of interest that most studies deal with the investigation of the SDF-1α-mediated induction of HSPC migration concomitantly with the decipherment of signal transduction cascades engaged by the SDF-1α receptor CXCR4. By contrast, considerably less is known about factors, conditions, and mechanisms that modulate the SDF-1α induced HSPC migration. Here we will give an overview about our research dealing with this topic and will show that the SDF-1α mediated migratory activity of cultured HSPCs strongly depends on the cytokines/cytokine combinations being used for HSPC cultivation. In fact, the removal of only one factor from a cytokine cocktail, which give rise to highly SDF-1α susceptible HSPCs, will yield in cells, which migratory activity is inhibited by SDF-1α. Additionally, we will also summarize our results concerning factors that might act as stop-signals for the SDF-1α induced migration of HSPCs, which may play a role in the termination of HSPC migration.
Extravasation has been described as a rate-limiting step in the process of hematogeneous metastasis formation. Thereby, transendothelial migration of tumor cells consists of a complex series of events involving multiple cell-cell and cell-matrix interactions. 3D-extravasation assays are valuable tools for the identification of genes, which are the key players at switchboards of the intracellular signaling pathways. In consequence, the combination of 3D-modeling and whole genome expression analysis lead to unravel molecular parameters which descripe distinct clinical phenotypes of cancer and therefore, work as prognosticators, predictors of therapy and new therapy targets.
