Automated real-time measurement of chemotactic cell motility.

We have developed a novel method, (ECIS/taxis), for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment, and their positions are monitored using the electric cell-substrate impedance sensor technology to measure the impedance change at a target electrode, that is lithographed onto the substrate, as the cells arrive at the target. In the studies reported here, Dictyostelium discoideum was used as a prototypical, motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose offolate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to the delayed arrival of cells at the target electrode. The multi-well assay configuration allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement.     

Automated topographical cell proliferation analysis

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.     

Automated characterization of cell shape changes during amoeboid motility by skeletonization

Background  

The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility.     

A new in vitro assay for cell motility and proliferation

We have described and characterized a new micropore membrane assay for migration and proliferation of cells of various tumourigenic potential. The assay was developed to facilitate analysis of some aspects of cancer invasion and metastasis. Tumorigenic and non-tumorigenic C3H/10 T 1/2 cells grow in and migrate out of a culture chamber during a 1-11 day period, the shorter periods are used for chambers with 6 micron thick polycarbonate membranes, the longer ones for 140 micron thick cellulose nitrate membranes. Cell growth within the chambers, in their micropore membranes and on the outside of the membranes, was assessed with microscopy, electronic cell counting, flow cytometry of propidium iodide (PI) stained cells, and 3H-thymidine [( 3H]TdR) incorporation. A complete retrieval of intact cells that have traversed the membraneous chamber wall is possible, and these cells can be recultured or used in other studies. The tumorigenic cells had a steeper growth curve in vitro than the non transformed cells, but the relative sizes of the emigrated subpopulations were not significantly different. The subpopulation of tumorigenic cells that emigrated spontaneously from the chambers was less able than the subpopulation retained to populate secondary chamber cultures, suggesting that the clonogenic (stem) tumour cells are 'slow movers'.     

A new in vitro assay for cell motility and proliferation

Abstract. We have described and characterized a new micropore membrane assay for migration and proliferation of cells of various tumourigenic potential. The assay was developed to facilitate analysis of some aspects of cancer invasion and metastasis. Tumorigenic and non-tumorigenic C3H/10 T1/2 cells grow in and migrate out of a culture chamber during a 1–11 day period, the shorter periods are used for chambers with 6 μ m thick polycarbonate membranes, the longer ones for 140 μ m thick cellulose nitrate membranes. Cell growth within the chambers, in their micropore membranes and on the outside of the membranes, was assessed with microscopy, electronic cell counting, flow cytometry of propidium iodide (PI) stained cells, and ³H-thymidine ([³H]TdR) incorporation. A complete retrieval of intact cells that have traversed the membraneous chamber wall is possible, and these cells can be recultured or used in other studies. The tumorigenic cells had a steeper growth curve in vitro than the non transformed cells, but the relative sizes of the emigrated subpopulations were not significantly different. The subpopulation of tumorigenic cells that emigrated spontaneously from the chambers was less able than the subpopulation retained to populate secondary chamber cultures, suggesting that the clonogenic (stem) tumour cells are 'slow movers'.     

99mTc-HMPAO labelling inhibits cell motility and cell proliferation and induces apoptosis of NC-NC cells

(99m)Tc-hexamethyl-propylenamine-oxime ((99m)Tc-HMPAO)-labelled leukocytes have been used in standard diagnostic procedures for the detection of infection and inflammation. Although some investigators have already pointed out that labelling of leukocytes with (99m)Tc-HMPAO has detrimental effects on the cells, still very little is known regarding the effects of ionizing radiation on lymphocyte function. The effects of (99m)Tc-HMPAO-labelling on lymphocyte adhesion, proliferation, mitotic index, migration and apoptosis were evaluated. The lymphoblastoid cell line NC-NC was used as the lymphocyte population. (99m)Tc-HMPAO-labelling decreased cell adhesion, proliferation, mitotic index and motility, whereas it induced apoptosis and cell-cycle arrest. The rate of decrease in cell proliferation was up to 70% (P<0.001) by day 4 after labelling. (99m)Tc-HMPAO-labelling led a 35% decrease (P<0.001) in adhesion ability of the cells on fibronectin at 16h. Using the Boyden chamber motility assay, it was shown that both spontaneous and monocyte chemotactic protein (MCP-1)-induced lymphocyte motility were strongly reduced by (99m)Tc-HMPAO-labelling. The decrease in motility was approximately five-fold (P<0.05). In addition, a 12-fold increase (P<0.05) was observed in apoptosis of the (99m)Tc-HMPAO-treated cells compared with control cells. Besides, it was shown that cell-cycle arrest was induced starting from the 3rd day after treatment with (99m)Tc-HMPAO. Our observations indicate that (99m)Tc-HMPAO-labelling has damaging effects on lymphocyte function including cell adhesion, proliferation, mitotic index, motility and cell cycle under in vitro conditions.     

Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts

Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle.     

Regulation of cell proliferation by autocrine motility factor/phosphoglucose isomerase signaling

Autocrine motility factor (AMF)/phosphoglucose isomerase (PGI; EC 5.3.1.9) is a housekeeping cytosolic enzyme that plays a key role in both glycolysis and gluconeogenesis pathways. AMF/PGI is also a multifunctional protein that displays cytokine properties, eliciting mitogenic, motogenic, and differentiation activities, and has been implicated in tumor progression and metastasis. Because little is known about AMF/PGI-dependent signaling in general and during tumorigenesis in particular, we sought to study its effect on the cell cycle. To elucidate the functional role of PGI, we stably transfected its cDNA into NIH/3T3 and BALB/c 3T3-A31 fibroblasts. Ectopic overexpression of PGI results in the acquisition of a transformed phenotype associated with an acceleration of G1 to S cell cycle transition. These were manifested by up-regulation of cyclin D1 expression and cyclin-dependent kinase activity and down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. The reduced p27Kip1 protein expression level in PGI-overexpressing cells could be restored to control levels by treatment with proteasome inhibitor. PGI-overexpressing cells also exhibited elevated expression of Skp2 involved in p27Kip1 ubiquitination and elevation in the levels of retinoblastoma protein hyperphosphorylation. Thus, we may conclude that the overexpression of AMF/PGI enhances cell proliferation together with up-regulation of cyclin/cyclin-dependent kinase activities and down-regulation of p27Kip1, whereas the induction of 3T3 fibroblast transformation by PGI is regulated by the retinoblastoma protein pathway.     

Gastrin inhibits motility,decreases cell death levels and increases proliferation in human glioblastoma cell lines

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 373–382, 1998     

Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement

Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.     

Adiponectin and leptin exert antagonizing effects on proliferation and motility of papillary thyroid cancer cell lines

Journal of Physiology and Biochemistry - Adiponectin (Acrp30) and leptin, adipokines produced and secreted mainly by the adipose tissue, are involved in human carcinogenesis. Thyroid carcinomas are...     

Simultaneous measurement of sensor-protein dynamics and motility of a single cell by on-chip microcultivation system

Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation. We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations. Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium. However, it took more than three generations for recovering the localization after the removal of aspartate from the medium. Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell. The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information.     

Automated measurement of ciliary beat frequency

Measurements of ciliary beat frequency using video images are dependent on observer interpretation. To obtain objective estimates of ciliary beat frequency from video-image sequences, a computer-based method was developed. Regions of interest of video-image sequences were selected and digitized. Variations in numerical values representing light intensity resulting from cilia beating were extracted and analyzed using autocorrelation techniques. The ciliary beat frequencies obtained for 14 in vitro experiments on ciliated cells or epithelium from the frog palate (Rana catesbeiana) over the range of frequencies 2-25 Hz correlated well with independent observer measurements (r = 0.979). The addition of such computer-based methods to video observer-based systems allows more objective and efficient determinations of ciliary beat frequency.