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PubMed1994

Characterization of oligomannoside binding to the surface of murine melanoma cells. Potential relationship to oligomannoside-initiated cell spreading.

The spreading of murine melanoma cells on unglycosylated laminin is initiated by soluble glycosylated laminins containing oligomannosides, or by mannan, and is inhibited by mannose (Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3356-3366). In the present study, melanoma cell recognition of oligomannosides was explored by several different methods. Comparison of cell spreading, initiated by related branched oligomannosides, showed that micromolar concentrations of Man6 and Man9 were able to restore cell spreading maximally, whereas Man3 was ineffective. Man9 bound to the melanoma cells in a bimodal manner at micromolar concentrations, reaching saturation, whereas Man3 showed linear binding in the same and higher ranges. Comparison of Man9 binding with Man9-initiated cell spreading demonstrated that maximal spreading occurred at approximately half-saturation. Competition experiments showed that mannose or mannan effectively impaired Man9 binding to the cells, whereas galactose or fucose was ineffective. Mannan-conjugated fluorescent beads bound in a diffuse pattern to the surface of melanoma cells, whereas underivatized beads did not bind. The distribution of laminin-binding beta 1 integrin on the cell surface was compared with surface mannan binding. The beta 1 integrin staining pattern did not match the mannan staining pattern either in attached, nonspread cells or in attached, spread cells. The composite results support a model in which occupancy of both an integrin and a cell surface lectin is required for murine melanoma cells to spread on glycosylated laminin.

Animals↗
PubMed1975

A comparison of the inhibition of leucocyte migration and monocyte spreading as in vitro assays for tuberculin hypersensitivity in man.

The ability of leucocyte migration inhibition and monocyte spreading inhibition test to detect tuberculin hypersensitivity was compared in the same twelve Mantoux-negative and fifteen Mantoux-positive persons. Tuberculin hypersensitivity expressed in vitro as migration or spreading inhibition, induced by 100 mug of PPD/ml, was assessed after 2 and 24, or 4 and 20 hr of incubation. A significant difference was found between negative and positive persons by migration inhibition at the early interval and by spreading inhibition at both intervals. When the two tests were compared on the basis of individual results, monocyte spreading inhibition appeared more discriminating (fewer results in the group of positive persons overlapped with those found among negative persons). Results of the monocyte spreading inhibition test correlated well with cutaneous reactions at both incubation intervals, while with migration inhibition the correlation was not so well expressed at either interval. Furthermore, a given change in skin reactivity of tuberculin-positive persons was reflected better in spreading inhibition than in migration inhibition indices. We conclude that the method of monocyte spreading inhibition compares favourably with the method of leucocyte migration inhibition, and it seems to be a suitable in vitro test for detection of tuberculin hypersensitivity in man.

Cell Migration Inhibition↗
PubMed1993

Ehrlich ascites tumour cell spreading is induced upon contact with immobilized isolated fibroblast plasma membranes and fibronectin.

Ehrlich ascites tumour (EAT) cells normally growing in suspension, adhere and spread upon contact with fibroblasts. Changes in EAT cell morphology and adhesiveness induced in contact with purified fibroblast plasma membranes, fibroblast extracellular matrix and fibronectin were investigated. EAT cells adhered readily to glass covered with fibroblast extracellular matrix or fibronectin, but they did not spread. Immobilization of fibroblast plasma membranes or fibronectin on nitrocellulose restored EAT cell ability to spread. Tumour cells spread on immobilized plasma membranes or fibronectin maintained a "fried egg" morphology, while these spread on living fibroblasts were rather elongated. EAT cells preincubated with anti-fibronectin serum lost their ability to adhere to any of the tested substrata: living and fixed fibroblasts, fibroblast extracellular matrix, nitrocellulose with immobilized fibroblast plasma membranes or fibronectin. When anti-fibronectin serum was applied only to these substrata and it was washed out before EAT cell plating, EAT cell adhesion and spreading were not significantly changed. Extraction of extracellular matrix components from immobilized fibroblast plasma membranes did not influence EAT adhesion and spreading. The results are discussed with reference to the specific role of fibronectin in the growth of EAT cells as a solid tumour or suspension.

Animals↗
PubMed1996

[The spread of contrast medium in the epidural space and the analgesic area in epidural anesthesia in elderly patients].

We evaluated the relationship between the spread of contrast medium in epidural space and the analgesia area in epidural anesthesia in elderly patients. The spread of contrast medium was examined radiographically after the injection of iotrolan 5 ml through the epidural catheter and the analgesia area was examined by pin prick following the epidural injection of the same volume of 1% lidocaine. In lumbar group (group L) (n = 10), the radiographic spread was 10.4 (4-17) segments (mean, minimum-maximum) and the analgesic area was 8.5 (4-15) dermatomes. In cervical or thoracic group (group C/T) (n = 10), the radiographic spread was 12.4 (8-17) segments and the analgesic area was 10.4 (6-15) dermatomes. The dose of local anesthetics required to get analgesia per. one dermatome was 0.7 (0.3-1.3) ml in group L and 0.5 (0.3-0.8) ml in group C/T. The spread of contrast medium correlated well with the analgesic area in both groups (group L:Y = 0.79X + 0.31, r = 0.92, group C/ T:Y = 0.80X + 0.52, r = 0.79). The mean dose of local anesthetics required for analgesia was smaller than that of previous reports although individual variations were large. We conclude that the spread of contrast medium is useful for predicting the spread of epidural block.

Aged↗
PubMed1996

Rho is a negative regulator of human monocyte spreading.

Monocyte spreading is a sequential series of events including cell flattening, formation of new actin structures and focal adhesions, as well as development of cytoplasmic projections. To investigate the involvement of the GTP-binding protein Rho in spreading, we treated human blood monocytes or PMA-stimulated U937 or THP1 cells with Clostridium botulinum C3-transferase (C3), which ADP-ribosylates and inactivates Rho in intact cells. The C3 treatment caused 1) a four- to fivefold increase in the number of THP1 cells that spread on fibronectin within 24 h of PMA stimulation, 2) a greater area covered by the spread cells, and 3) accelerated and enhanced development of macrophage-like filopodial and pseudopodial projections. Similar results were obtained with PMA-stimulated U937 cells and human blood monocytes. Furthermore, cell staining revealed disorganization of subcortical actin in C3-treated THP1 cells, whereas circular actin formations at the substrate-attached part of the cells and vinculin-containing focal complexes/adhesions were unaffected. Finally, we found a decrease in membrane-associated RhoA in normal spreading THP1 cells, which suggests endogenous inactivation of Rho and might provide an explanation for the acceleration of spreading caused by the C3-transferase. In conclusion, these results indicate that active Rho is an important, negative regulator of human monocyte spreading by maintaining cell tension and cortical actin organization.

ADP Ribose Transferases↗
PubMed1998

Oxidant stress incites spreading of macrophages via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase.

Cultured macrophages exhibit spreading in response to external stimuli. It is relevant to in vivo morphologic changes of macrophages during extravasation, migration, and differentiation. The present study was performed to elucidate molecular mechanisms that regulate spreading of macrophages. Redox is a crucial factor that modulates a wide range of cell function. We found that macrophages undergo spreading in response to oxidant stress caused by hydrogen peroxide or an oxidant generating agent menadione. To identify signaling pathways involved, a role of mitogen-activated protein (MAP) kinases was investigated. Western blot analysis showed that treatment of macrophages with menadione rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK1, ERK2) and p38 MAP kinase, but not c-Jun N-terminal kinase (JNK). Pharmacologic inhibition of either ERK or p38 activation blunted the macrophage spreading. Similarly, transfection with dominant-negative mutants of ERKs or a mutant p38 significantly suppressed the oxidant-triggered spreading. ERKs and p38 are known to activate serum response element (SRE) via phosphorylation of the ternary complex factor Elk-1. To further identify downstream events, we focused on a role of SRE. Stimulation of macrophages with menadione induced activation of SRE. Intervention in the SRE activation by a dominant-negative mutant of Elk-1 inhibited the menadione-induced spreading. These results suggest that oxygen radical metabolites, the well-known mediators for tissue injury, incite spreading of macrophages via the MAP kinase-SRE signaling pathways.

Animals↗
PubMed1996

Effects of antimigraine drugs on retinal spreading depression.

It has been suggested that spreading depression may play a role in triggering classical migraine. In this study the retinal spreading depression was used as a pharmacological tool to test the neuronal effects of several common antimigraine drugs. As the chicken retina is void of any blood vessels the observed effects must be of pure neuronal origin. It is shown that propranolol, sumatriptan, methysergide, paracetamol and acetylsalicyclic acid decrease the propagation velocity of retinal spreading depression waves, accelerate the recovery of the optical and electrical signal and reduce the amplitude of the negative potential shift, concomitant with the spreading depression. Barbiturate increases the spreading velocity, and the amplitude of the potential shift. Ergotamine, clonidine, lisuride and iprazochrome have no significant influence on retinal spreading depression.

Acetaminophen↗
PubMed1993

Rapid increase of BDNF mRNA levels in cortical neurons following spreading depression: regulation by glutamatergic mechanisms independent of seizure activity.

Levels of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and the tyrosine kinase receptors trkB and trkC have been studied using in situ hybridization in the rat brain after topical application of KCl to the cortical surface (which induces spreading depression). Repeated episodes of spreading depression during 2 h caused a rapid and marked increase of BDNF mRNA levels in deep and, in particular, superficial cortical layers of the ipsilateral hemisphere (to 213 and 417% of control, respectively). Maximal levels were reached within 2 h after the cessation of spreading depression and at 24 h BDNF mRNA expression had returned to control values. Levels of BDNF mRNA were unaffected in the hippocampus, in areas outside the cerebral cortex and in the contralateral hemisphere. Furthermore, no change of the expression of mRNA for NGF, NT-3, trkC or the full length trkB receptor was detected at any time point. However, at 2 h after spreading depression there was an increased level (150% of control) in superficial cortical layers of mRNA hybridizing to an oligonucleotide probe detecting both truncated receptors lacking the tyrosine kinase domain and full length trkB receptors. Also one single episode of spreading depression gave rise to a significant increase of cortical BDNF mRNA levels (to 207% of control), which was attenuated (by 61%) after administration of the competitive NMDA receptor antagonist CGS 19755. The results provide evidence that mild brain insults associated with glutamate release and elevated intracellular calcium, such as spreading depression, also in the absence of seizure activity can lead to activation of the BDNF gene in cortical neurons.

Analysis of Variance↗
PubMed2026

Reconstructing the early spatial spread of pandemic respiratory viruses in the United States.

Understanding the geographic spread of emerging respiratory viruses is critical for pandemic preparedness, yet the early spatiotemporal dynamics of the 2009 H1N1 pandemic influenza and severe acute respiratory syndrome coronavirus 2 in the United States remain unclear. While mobility and genomic data have revealed important aspects of pandemic spatial spread, several key questions remain: Did the two pandemics follow similar spatial transmission routes? How rapidly did they spread across the United States? What role did stochastic processes play in early spatial transmission? To address these questions, we integrated high-resolution disease data with a robust, data-efficient inference framework combining air travel, commuting flows, and pathogen superspreading potentials to reconstruct their spatial spread across US metropolitan areas. The two pandemics exhibited distinct transmission pathways across locations; however, both pandemics established local circulation in most metropolitan areas within weeks, driven by several shared transmission hubs. Early spatial spread was more strongly associated with air travel than with commuting, though stochastic dynamics introduced substantial uncertainty in transmission routes, creating challenges for timely detection and control. Simulations indicate that broad wastewater surveillance coverage beyond top transmission hubs coupled with effective infection control may slow initial spatial expansion. Our findings highlight the rapid, stochastic spread of pandemic respiratory pathogens and the difficulties of early outbreak containment.

Humans↗
PubMed2005

Cortical spreading depression and peri-infarct depolarization in acutely injured human cerebral cortex.

Electrocorticographic (ECoG) activity was recorded for up to 129 h from 12 acutely brain-injured human patients using six platinum electrodes placed near foci of damaged cortical tissue. The method probes ECoG activity in the immediate vicinity of the injured cortex and in adjacent supposedly healthy tissue. Six out of twelve patients displayed a total of 73 spontaneous episodes of spreading depression of the ECoG. Of the remaining 6 patients 1 displayed an episode of synchronous depression of ECoG during surgery. Using the same electrodes we also measured the slow potential changes (SPC) (0.005-0.05 Hz) to test the hypothesis that the ECoG depressions were identical to Leao's cortical spreading depression (CSD), and to be able to record peri-infarct depolarisations (PIDs) in electrically 'silent' cortical tissue. Changes in the SPC indicate depolarization of brain tissue. For the analysis, the SPCs were enhanced by calculating the time integral of the ECoG signal. Spreading ECoG depressions were accompanied at every single recording site by stereotyped SPCs, which spread across the cortical mantle at 3.3 (0.41-10) mm/min (median, range), i.e. at the same speed of spread as the depression of the ECoG activity. The amplitude of the SPCs was 0.06-3 mV. In 4 out of 6 patients the ECoG recovered spontaneously. In 2 patients we subsequently recorded recurrent SPCs, but without recovery of the initial ECoG background activity until 2-5 h later. This represents the first direct recording of PIDs in acutely injured human brain. Evidence from this and our previous study of 14 brain-injured patients suggests that CSDs in acute brain disorders occur at higher incidence in patients <30 years (83%) than above (33%). CSD was recorded in 4 out of 5 traumatic brain injury patients, and in 2 out of 7 patients with spontaneous haemorrhages. We conclude that the spreading ECoG depressions recorded in patients are identical to CSDs recorded in animal experiments. We furthermore provide direct electrophysiological evidence for the existence of PIDs and hence a penumbra in the human brain. We hypothesize that the depolarization events might contribute to tissue damage in acute disorders in the human brain.

Adult↗
PubMed1996

Characterization of endothelin receptors in the cerebral vasculature and their lack of effect on spreading depression.

The changes in cerebral blood flow that accompany spreading depression are well-described, as are parallel changes in cellular activity, with a wave of hyperemia followed by a prolonged oligemic phase. In this study, a cat model of the CBF changes associated with spreading depression and in vitro pharmacology were used to determine if there is a role for the powerful peptide vasoconstrictor endothelin in this response. For the pharmacological studies, the middle cerebral artery was harvested from cats postmortem. For the physiological studies, cats were anesthetized with halothane induction and alpha-chloralose (60 mg/kg, intraperitoneal loading; 20 mg/kg i.v. 2-h maintenance). CBF was monitored continuously in the parietal cortex using laser Doppler flowmetry (CBFLDF) after exposure of the dura mater. The in vitro work demonstrated that endothelin-1 (ET-1) mediates a strong and potent contraction of cerebral vessels. Both the selective ETA receptor antagonist FR139317 and the combined ETA and ETB receptor antagonist Bosentan caused a rightward shift of the concentration-response curve without attenuation of the maximum effect. The calculated pA2 values were 6.28 and 6.90, respectively. The slope did not differ from unity, suggesting that the ET-1-mediated contraction is evoked by a single population of ETA receptors, which were effectively antagonized by these compounds. Spreading depression was induced with a needle stick injury to the cortex. Local administration of the endothelin antagonists FR139317 (10 microM) and Bosentan (10 microM) did not affect resting blood flow in the cortex. Induction of spreading depression following local administration of FR139317 and Bosentan resulted in responses no different from those in control cortex. These data demonstrate that endothelin does not play a significant role in the vasoconstrictor portion of the CBF change seen in spreading depression, nor does it affect resting flow. Since it is widely held that spreading depression, or a very similar mechanism, underlies the aura phase of migraine, it may be suggested from these studies that endothelin antagonists are unlikely to be useful in migraine.

Animals↗
PubMed2005

Topiramate inhibits cortical spreading depression in rat and cat: impact in migraine aura.

Cortical spreading depression is thought to be the pathophysiological correlate of the neurological symptoms in migraine with aura. Topiramate is an anti-epileptic drug that is also used as a migraine preventive. Ion homeostasis and excitatory amino acid efflux are major components of cortical spreading depression; so given the broad range of actions of topiramate, we examined its effect on cortical spreading depression. Cortical spreading depression was elicited by a needle stick in the cortex in surgically prepared rats and cats; laser Doppler probes were used to measure the cerebral blood flow and a recording electrode was used to measure electrical nerve cell activity. Topiramate at 30 mg kg(-1) was able to inhibit regional cerebral blood flow changes and cortical depolarization and spreading depression in all rats, and in 8 of 13 cats. We conclude that topiramate may act on mechanisms involved in the initiation and propagation of spreading depression, and that these mechanisms may be similar to those involved in the therapeutic role.

Analysis of Variance↗
PubMed2004

Energy failure in astrocytes increases the vulnerability of neurons to spreading depression.

A neuroprotective role of astrocytes has been hypothesized, but the mechanism is debated and in vivo evidence is limited. To test this hypothesis, a sublethal stressor (spreading depression) and fluorocitrate (FC), a selective inhibitor of the astrocytic Krebs cycle, were used in urethane-anaesthetized adult rats. Neuronal damage was assessed 24 h after treatment with silver stain and immunoreactivity for a 72-kDa heat-shock protein. ATP levels and mitochondrial aconitase activity, a marker indicating exposure to reactive oxygen species, were measured after 4 and 24 h. Spreading depression alone did not affect ATP levels, mitochondrial aconitase activity, or induce neuronal injury in the cortex. Local or intraventricular injection of FC significantly decreased ATP levels and mitochondrial aconitase activity, but did not produce neuronal damage. In animals receiving injections of FC and then spreading depression, there was evidence of significant neuronal stress and damage. Isocitrate, which bypasses the metabolic inhibition produced by FC, prevented all of the changes seen after the combination of FC and spreading depression. One-hour pretreatment with dimethyl sulfoxide (a scavenger of hydroxyl radicals), deferoxamine (an iron chelator) or fructose-1,6-bisphosphate also blocked inactivation of mitochondrial aconitase, ATP depletion and the neuronal damage induced by FC and spreading depression. These experiments demonstrate that inhibition of the metabolism of astrocytes, with a decrease in ATP levels, will increase the susceptibility of neurons to the stress induced by spreading depression. The neuroprotective effects of dimethyl sulfoxide, deferoxamine and fructose-1,6-bisphosphate suggest that oxidative stress contributes to the neurotoxicity in this situation.

Aconitate Hydratase↗
PubMed2001

Ischaemia triggered by spreading neuronal activation is inhibited by vasodilators in rats.

It has been previously shown that spreading neuronal activation can generate a cortical spreading ischaemia (CSI) in rats. The purpose of the present study was to investigate whether vasodilators cause CSI to revert to a normal cortical spreading depression (CSD).A KCl-induced CSD travelled from an open cranial window to a closed window where the cortex was superfused with physiological artificial cerebrospinal fluid (ACSF). At the closed window, recordings revealed a short-lasting negative slow potential shift accompanied by a variable, small and short initial hypoperfusion followed by hyperaemia and then oligaemia. In contrast, spreading neuronal activation locally induced CSI at the closed window when ACSF contained a NO. synthase (NOS) inhibitor, N(G)-nitro-L-arginine, and an increased K+ concentration ([K+]ACSF). CSI was characterised by a sharp and prolonged initial cerebral blood flow decrease to 29 +/- 11 % of the baseline and a prolonged negative potential shift. Co-application of a NOá donor, S-nitroso-N-acetylpenicillamine, and NOS inhibitor with high [K+]ACSF re-established a short-lasting negative potential shift and spreading hyperaemia typical of CSD. Similarly, the NO.-independent vasodilator papaverine caused CSI to revert to a pattern characteristic of CSD. In acute rat brain slices, NOS inhibition and high [K+]ACSF did not prolong the negative slow potential shift compared to that induced by high [K+]ACSF alone. The data indicate that the delayed recovery of the slow potential was caused by vasoconstriction during application of high [K+]ACSF and a NOS inhibitor in vivo. This supports the possibility of a vicious circle: spreading neuronal activation induces vasoconstriction, and vasoconstriction prevents repolarisation during CSI. Speculatively, this pathogenetic process could be involved in migraine-induced stroke.

Animals↗
PubMed2002

Predictors of extensive subclinical spread in nonmelanoma skin cancer treated with Mohs micrographic surgery.

BACKGROUND: In nonmelanoma skin cancer, the clinically visible portion may represent a small fraction of microscopic tumor spread. Previous studies have examined individual risk factors for subclinical spread based on patient and tumor characteristics. However, these risk factors have not been prioritized or studied in combination. OBJECTIVE: To identify the most predictive risk factors for extensive subclinical tumor spread. DESIGN: Retrospective analysis of 1131 Mohs micrographic surgical cases. Variables analyzed included patient age, sex, and immune status and lesion size, location, histologic subtype, and recurrence. Logistic regression was applied to identify important combinations of tumor characteristics and to quantify relative odds of spread. SETTING: Academic referral center. PATIENTS: Consecutive sample of all referred patients treated by a single Mohs micrographic surgeon in a 3-year period. MAIN OUTCOME MEASURE: Number of Mohs micrographic surgical layers required to clear a tumor, with 3 or more layers defined as extensive subclinical spread. RESULTS: The highest-risk tumors, with odds ratios greater than 6.0, were basosquamous and morpheaform basal cell carcinoma (BCC) on the nose, morpheaform BCC on the cheek, and those with a preoperative size greater than 25 mm. Other important risk factors were recurrent and nodular BCC on the nose; location on the eyelid, temple, or ear helix; neck tumors and recurrent BCC in men; and tumor size greater than 10 mm. Patients younger than 35 years were at lower risk. Increasing age and immunocompromise were not significant predictors. CONCLUSION: Identification of lesions likely to exhibit extensive subclinical spread can help guide management to ensure complete tumor eradication and thereby reduce the risk of recurrence and its associated morbidity and cost.

Adult↗
PubMed1999

Spreading, proliferation, and differentiation of the epidermis after wounding a cichlid fish, Hemichromis bimaculatus.

A large superficial wound has been experimentally provoked in the cichlid fish Hemichromis bimaculatus to study the interactions between the epidermal cells and the substrate on which they spread, on the one hand, and the restoration of the subepidermal tissues and the epithelial-mesenchymal interactions preceding scale regeneration, on the other hand. The re-epithelialization process, e.g., migration, spreading, differentiation, and proliferation of the epidermal cells, has been followed step by step, using light, scanning and transmission electron microscopy, and tritiated thymidine incorporation, until complete reorganization of the healing epidermis. Wound healing is fast (500 microm/hr) and proceeds centripetally from the wound margins. The epidermal cells spread on a wound surface which is composed of two different matrices: the remains of basement membrane materials covering the scale-pockets, and collagen fibrils of cut dermal strips. Even though both matrices favour cell spreading and attachment, migrating cells show a different behaviour. The re-epithelialization of the wound follows an orderly sequence similar to amphibian and mammalian wound healing, i.e., a "leap frog" mechanism of cell locomotion involving three epidermal layers. The basal layer cells, which spread on the substrate, and the superficial layer cells which protect the epidermis, differentiate first. Whatever the type of substrate over which the epithelium spreads (basement membrane material or collagen fibrils), the epidermal basal layer cells differentiate as soon as they become attached. The incorporation of tritiated thymidine has revealed that there is no proliferation in the healing epidermis until after complete closure of the wound, but that the rapid re-epithelialization of the large surface requires the recruitment of epidermal cells at the wound margins. The present study offers new data on the dynamics of re-epithelialisation and on the resistance of cichlid skin to such wounds. It is also clearly shown that the epidermal basal layer cells differentiate rapidly, a step which is interpreted as the first stage of epithelial-mesenchymal interactions that will lead to scale regeneration.

Animals↗
PubMed1997

Altered in vitro spreading and cytoskeletal organization in human glioma cells by downregulation of urokinase receptor.

The interaction of urokinase-type plasminogen activator (uPA) with its cell-surface receptor (uPAR) is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that uPAR can act as an extracellular matrix receptor during cell adhesion. Recently, we showed that transfection of the human glioma cell line SNB19 with antisense uPAR resulted in downregulation of uPAR at both the mRNA and protein levels. In this study, we used SNB19 to determine how the presence or absence of uPAR promotes cell spreading and associated changes in cell morphology. Microscopic analysis of cell spreading revealed that antisense uPAR-transfected cells were larger, remained round, and did not spread efficiently over extracellular matrix substrate type IV collagen and fibronectin, unlike parental SNB19 cells, which were smaller and spindle shaped. Biochemical studies showed that antisense uPAR-transfected cells, in addition to not spreading, exhibited increased expression of alpha 3 beta 1 integrin but not alpha 5 beta 1 integrin. However, we could not find a change in the expression of extracellular matrix components or altered growth rate in these cells. Furthermore, despite the increased alpha 3 beta 1 integrin expression, antisense uPAR-transfected cells failed to form an organized actin cytoskeleton when plated on type IV collagen or fibronectin, unlike parental SNB19 cells, which displayed an organized cytoskeleton. These findings show that the absence of uPAR in human glioma cells leads to morphological changes associated with decreased spreading and a disorganized cytoskeleton resulting in altered cell morphology, suggesting that coordinated expression of uPAR and integrin may be involved in spreading of antisense uPAR-transfected glioma cells.

Cell Adhesion↗
PubMedDate not supplied

Quantification of VP22-GFP spread by direct fluorescence in 15 commonly used cell lines.

BACKGROUND: The intercellular transport property of VP22 chimeric proteins offers the opportunity for the improvement of gene therapy delivery systems. Since enhanced therapeutic effects of transduced genes already have been exemplified for chimeric proteins VP22-p53 and VP22-tk, we were interested in examining whether spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. METHODS: To study intercellular spread of VP22-GFP fusion proteins, 15 different mammalian cell lines were transfected with 200-2000 ng of VP22-GFP or GFP expression plasmids. Expression of VP22-GFP or GFP was monitored by fluorescence microscopy of live GFP fluorescence and direct FACS analysis. For selected cell lines, antibody detection of VP22-GFP spread was analysed by confocal microscopy as a control. RESULTS: Spread of VP22-GFP fusion proteins was detected in all 15 cell lines tested, and quantified by FACS analysis. Experimental conditions were found to be critical in the investigation of VP22-mediated intercellular spread. CONCLUSION: Results of our study indicate that spread of VP22 chimeric proteins is a general biological phenomenon not restricted to distinct tissues or species. Therefore, further evidence is provided that VP22-enhanced gene therapeutic effects may be obtained irrespective of the target organ/tissue to be addressed.

Animals↗