Virus-induced cell motility

Many viruses induce profound changes in cell metabolism and function. Here we show that vaccinia virus induces two distinct forms of cell movement. Virus-induced cell migration was demonstrated by an in vitro wound healing assay in which infected cells migrated independently into the wound area while uninfected cells remained relatively static. Time-lapse microscopy showed that the maximal rate of migration occurred between 9 and 12 h postinfection. Virus-induced cell migration was inhibited by preinactivation of viral particles with trioxsalen and UV light or by the addition of cycloheximide but not by addition of cytosine arabinoside or rifampin. The expression of early viral genes is therefore necessary and sufficient to induce cell migration. Following migration, infected cells developed projections up to 160 microm in length which had growth-cone-like structures and were frequently branched. Time-lapse video microscopy showed that these projections were formed by extension and condensation of lamellipodia from the cell body. Formation of extensions was dependent on late gene expression but not the production of intracellular enveloped (IEV) particles. The requirements for virus-induced cell migration and for the formation of extensions therefore differ from each other and are distinct from the polymerization of actin tails on IEV particles. These data show that poxviruses encode genes which control different aspects of cell motility and thus represent a useful model system to study and dissect 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.     

The role of laminin-5 in TGF alpha/EGF-mediated corneal epithelial cell motility

Fish and amphibian oocytes provide excellent experimental systems for both biochemical and cytological analyses of regulatory mechanisms of meiotic maturation and arrest. Recent work shows that despite the adoption of common players, such as maturation-promoting factor (MPF), c-mos proto-oncogene product (Mos), and mitogen-activated protein kinase (MAPK), there is clear species-specificity in the mechanisms, probably due to the difference in the states of inactive MPF in immature oocytes. However, it has also been revealed that the mechanisms controlling meiotic maturation and arrest include ubiquitous pathways; The translational activation of masked mRNAs encoding Mos and cyclin B for initiating maturation and the Mos-MAPK pathway for maintaining metaphase arrest.     

Gliding motility: an efficient mechanism for cell penetration

An important group of animal and human pathogens, belonging to the phylum Apicomplexa, employs a novel form of motility, known as gliding, to move on solid substrates and to enter host cells. Gliding is dependent on the parasite cytoskeleton and involves a conserved family of secretory adhesins.     

Patterns and control of cell motility in the Xenopus gastrula

By comparing cells with respect to several motility-related properties and the ability to migrate on fibronectin, three cell types can be distinguished in the Xenopus gastrula. These occur in a distinct spatial pattern, thus defining three motility domains which do not correspond to the prospective germ layers. Migratory behavior is confined to a region encompassing the anterior mesoderm and endoderm. When stationary animal cap cells are induced to migrate by treatment with activin, cells become adhesive at low concentrations of fibronectin, show polarized protrusive activity, and form lamellipodia. Adhesion and polarization, but not lamellipodia formation, are mimicked by the immediate early response gene Mix.1. Goosecoid, another immediate early gene, is without effect when expressed alone in animal cap cells, but it acts synergistically with Mix.1 in the control of adhesion, and antagonistically in the polarization of protrusive activity. bFGF also induces migration, lamellipodia formation and polarization in animal cap cells, but has no effect on adhesion. By the various treatments of animal cap cells, new combinations of motile properties can be generated, yielding cell types which are not found in the embryo.     

Molecular control of vascular smooth muscle cell differentiation

Changes in the differentiated state of the vascular smooth muscle cell (SMC) including enhanced growth responsiveness, altered lipid metabolism, and increased matrix production are known to play a key role in development of atherosclerotic disease. As such, there has been extensive interest in understanding the molecular mechanisms and factors that regulate differentiation of vascular SMC, and how this regulation might be disrupted in vascular disease. Key questions include determination of mechanisms that control the coordinate expression of genes required for the differentiated function of the smooth muscle cell, and determination as to how these regulatory processes are influenced by local environmental cues known to be important to control of smooth muscle differentiation. Of particular interest, a number of common cis regulatory elements including highly conserved CArG [CC(A/T)6GG] motifs or CArG-like motifs and a TGF beta control element have been identified in the promoters of virtually all smooth muscle differentiation marker genes characterized to date including smooth muscle alpha-actin, smooth muscle myosin heavy chain, telokin, and SM22 alpha and shown to be required for expression of these genes both in vivo and in vitro. In addition, studies have identified a number of trans factors that interact with these cis elements, and shown how the expression or activity of these factors is modified by local environmental cues such as contractile agonists that are known to influence differentiation of smooth muscle.     

Integrins, angiogenesis and vascular cell survival

The interactions between integrins and the extracellular matrix have been identified as important regulators of vascular cell survival, proliferation and invasion during the complex process of blood vessel formation by angiogenesis.     

Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility

The azo-dye, Fast Blue (FB), initially employed for retrograde neuronal tracing is increasingly used in cell invasion and migration studies to detect living cells in monolayer and glioma tumor cell spheroid models. As yet, it is assumed that a cell stained with a tracker dye retains the characteristics of the original cell. The following experiments compared the adhesion, migration and proliferation properties of the cell lines U373 and GaMG with and without FB staining. FB staining reduced cell adhesion (P < 0.01) and proliferative activity (P < 0.01) and also had a significant inhibitory effect on cell migration (P < 0.001). From the results presented it follows that FB staining markedly influences basic cell characteristics.     

Estrogen receptor mediated inhibition of cancer cell invasion and motility: an overview

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER alpha was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.     

The effect of vascular endothelial cell growth factor on survival of skin flap in rats

In order to study the effect of vascular endothelial cell growth factor (VEGF) on the survival of skin flap 30 SD rats were used. A randomized flap measuring 7.5 cm x 3.0 cm was created on the back of each SD rat. The treatment group (n = 10) received VEGF 40 ng/flap by subcutaneous injection with microinjector during and 24 hours after operation. The control groups received heparin 16 U/flap (n = 10) or normal saline 800 microliters/flap (n = 10). After operation, on the 3rd and 11th day, the survival rate of the skin flaps and the dermovascular density of each flap were investigated by histological and histo-morphometrical examination. The results showed that there was no significant difference in the survival rate between the treatment group and the controls on the 3rd day after operation, while on the 11th day, there was a significant difference between them, and the survival rate was much higher in the treatment group. Besides, dermovascular density was much more increased in the treatment group than that in the controls, especially in the distal 1/3 of the flap (P < 0.02). The conclusion was that VEGF could accelerate the vascularization of the flap and enhance the survival rate of the flap.     

Control of vascular smooth muscle cell growth in fowl

In adult domestic fowl, angiotensin (ANG) receptors are present in the vascular smooth muscles (VSM) and in the endothelium, mediating vasorelaxation via endothelium-derived relaxing factor/cGMP. ANG II-induced relaxation is minor in chicks and becomes more marked as they mature but diminishes in adult birds, whereas ANG II neither relaxes nor contracts endothelium-denuded aortae from mature chickens. The present study examines in cultured fowl aortic SM cells whether (1) ANG II stimulates or inhibits VSM cell growth and, if so, whether this growth-stimulatory or -inhibitory effect changes with maturation/aging, and (2) S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, and cGMP attenuate the basal or stimulated VSM cell growth. [Asp1, Val5]ANG II (native fowl ANG II, 10(-6) M) markedly increased (increase from vehicle control, 226.5%; P < 0.01) [3H]thymidine (Thd) incorporation into DNA of quiescent VSM cells (first subculture) from 6-week-old chicks. This growth-stimulating effect was reduced with age (41.4, 29.6, and 3.2% at 9, 19, and 43 weeks of age, respectively). In contrast, platelet-derived growth factor (PDGF, 20 ng/ml) increased [3H]Thd incorporation similarly in chicks, pullets, and hens. Furthermore, ANG II significantly (45.9%, P < 0.01) attenuated the growth-promoting effect of fetal calf serum in cultured VSM cells from 6-week-old chicks. This inhibitory effect also decreased in older birds. ANG II showed neither a growth-stimulatory nor -inhibitory effect in cultured neointimal cells. SNAP attenuated dose dependently (20-60 microM) the basal and PDGF-induced VSM cell growth, whereas cGMP inhibited basal growth only at a high dose (100 microM). These results indicate that in fowl VSM cells, ANG II is mitogenic and antimitogenic in chicks but not in mature birds, suggesting that phenotypic modulation occurs in the ANG receptors/signaling mechanism with maturation/age or in neointimal cells, whereas the mitogenic mechanism via PDGF remains in both young and mature birds.     

A dynamic model of outer hair cell motility including intracellular and extracellular fluid viscosity

The deformation response of a guinea pig outer hair cell is modeled for mechanical and electrical stimulation up to 25 kHz. The analysis uses a Fourier series technique for a finite length cell surrounded internally and externally by a much larger continuum of viscous fluid. The analytical solution predicts that outer hair cell length changes occur due to applied mechanical or electrical stimulation without significant resonance, characteristic of a highly damped system. The deformation is found to have little attenuation up to a corner frequency of about 2 kHz for long cells and 10 kHz for short cells, in agreement with published experimental results. For electrical loading of 1 mV across the lateral cell wall, deformation for short cells is calculated to be greater than 1 nm for frequencies up to 20 kHz. These results support the proposition that in vivo the outer hair cell modifies the character of basilar membrane deformation on a cycle-by-cycle basis. An estimate of the capability of the cell to supply energy to the basilar membrane is given based on published values of outer hair cell material properties.     

The Rho GTPases in macrophage motility and chemotaxis

The present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin- in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imida zole . HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 microM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.     

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion

Genes that encode enzymes that convert inactive prodrugs into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a bystander assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.     

Microvascular endothelial cell control of peripheral vascular resistance during sepsis

OBJECTIVE: To determine the endothelial-dependent control of decreased peripheral vascular resistance in skeletal muscle microvessels during evolving sepsis. MATERIALS AND INTERVENTIONS: Acute (4 hours, n=7), established (24 hours, n=7), or chronic (72 hours, n=8) infection was induced in Sprague-Dawley rats (150-175 g) by injecting Escherichia coli and Bacteroides fragilis (1 x 10(9) colony-forming units for both) into a subcutaneous sponge. Control animals were injected with an isotonic sodium chloride solution and analyzed at the same time points: (n=6-8 per group). Dilation in response to the topically applied endothelial-dependent agonist acetylcholine (ACH) (1 x 10(-9) to 1 x 10(-5) mol/L) was measured in inflow first-order (A1) and precapillary fourth-order (A4) arterioles in cremaster muscle in vivo with videomicroscopy. Acetylcholine dose-response curves were used to determine vascular reactivity by calculating the concentration of ACH necessary to elicit 50% of the maximal dilator response. MAIN OUTCOME MEASURES: In vivo reactivity of striated muscle microvessels to the dilation agonist ACH during acute, established, and chronic infection. RESULTS: A1 vessels were unresponsive to all doses of ACH at all time points. A4 vessels showed an increased dilator response during short-term treatment, which deteriorated over time to depressed dilation during chronic infection. CONCLUSIONS: Precapillary A4 vessels have increased dilator reactivity during early sepsis, which progresses to depressed levels with chronic infection. A1 microvessels remain dilated and are not substantially influenced by endothelial dilator mechanisms initiated by ACH. Maximum dilation of the large A1 vessels appears to contribute to the decrease in peripheral vascular resistance noted during systemic infection.