Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

cAMP response element binding protein H mediates fenofibrate-induced suppression of hepatic lipogenesis.

AIMS/HYPOTHESIS: Fenofibrate is a drug used to treat hyperlipidaemia that works by inhibiting hepatic triacylglycerol synthesis. Sterol regulatory element binding protein-1c (SREBP-1c) is a major regulator of the expression of genes involved in hepatic triacylglycerol synthesis. In addition, endoplasmic reticulum (ER)-bound transcription factor families are involved in the control of various metabolic pathways. Here, we show a novel function for an ER-bound transcription factor, cAMP response element binding protein H (CREBH), in fenofibrate-mediated inhibition of hepatic lipogenesis. METHODS: The effects of fenofibrate and adenovirus-mediated Crebh (also known as Creb313) overexpression (Ad-Crebh) on hepatic SREBP-1c production and lipogenesis in vitro and in vivo were investigated. We also examined whether downregulation of endogenous hepatic Crebh by small interfering (si)RNA restores the fenofibrate effect on hepatic lipogenesis and SREBP-1c production. Finally, we examined the mechanism by which CREBH inhibits hepatic SREBP-1c production. RESULTS: Fasting and fenofibrate treatment induced CREBH production and decreased SREBP-1c levels. Indeed, Ad-Crebh inhibited insulin- and liver X receptor agonist TO901317-induced Srebp-1c (also known as Srebf1) mRNA expression in cultured hepatocytes. Moreover, increased production of CREBH in the liver of mice following tail-vein injection of Ad-Crebh inhibited high-fat diet-induced hepatic steatosis through inhibition of Srebp-1c expression. The inhibition of endogenous Crebh expression by siRNA restored fenofibrate-induced suppression of Srebp-1c expression and hepatic lipid accumulation both in vitro and in vivo. CONCLUSIONS/INTERPRETATION: These results show that fenofibrate decreases hepatic lipid synthesis through induction of CREBH. This study suggests CREBH as a novel negative regulator of SREBP-1c production and hepatic lipogenesis.

Redox compartmentalization and cellular stress.

Mammalian cells are highly organized to optimize function. For instance, oxidative energy-producing processes in mitochondria are sequestered away from plasma membrane redox signalling complexes and also from nuclear DNA, which is subject to oxidant-induced mutation. Proteins are unique among macromolecules in having reversible oxidizable elements, 'sulphur switches', which support dynamic regulation of structure and function. Accumulating evidence shows that redox signalling and control systems are maintained under kinetically limited steady states, which are highly displaced from redox equilibrium and distinct among organelles. Mitochondria are most reducing and susceptible to oxidation under stressed conditions, while nuclei are also reducing but relatively resistant to oxidation. Within compartments, the glutathione and thioredoxin systems serve parallel and non-redundant functions to maintain the dynamic redox balance of subsets of protein cysteines, which function in redox signalling and control. This organization allows cells to be poised to respond to cell stress but also creates sites of vulnerability. Importantly, disruption of redox organization is a common basis for disease. Research tools are becoming available to elucidate details of subcellular redox organization, and this development highlights an opportunity for a new generation of targeted antioxidants to enhance and restore redox signalling and control in disease prevention.

Novel selective dyeing method for chrysotile asbestos detection in concrete materials.

There are a tremendous number of asbestos-containing buildings without any surveys on the presence of asbestos because of the difficulty to detect asbestos in building materials simply and quickly, although a great deal of worldwide effort was put into removing asbestos of which inhalation causes serious diseases. In this study, we newly developed a simple dyeing method to detect chrysotile asbestos, the most commonly used type of asbestos, in asbestos-cement composite materials using magnesium-chelating organic dyes. As an essential process for selective dyeing of chrysotile asbestos, special pretreatment with a calcium-chelating agent was developed to prevent the dyes from reacting with calcium, which is the major component of concrete materials. Our developed selective dyeing method was shown to possess sufficient sensitivity for detecting chrysotile asbestos in an amount greater than 0.1 mass% in concrete specimens, and there was an approximately linear relationship between the area fraction of dyed spots and the mass fraction of chrysotile asbestos. Our results may provide a basis for further development of a simple on-site detection method for chrysotile asbestos in building materials and may facilitate the progress of control and removal of asbestos in the environment.

Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress.

Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

A novel role for the urokinase-type plasminogen activator receptor in apoptosis of malignant gliomas.

Glioblastomas express more urokinase-type plasminogen activator receptor (uPAR) than do low-grade gliomas and normal brain tissue. We previously showed that downregulation of uPAR through the transfection of SNB19 cells with an antisense cDNA construct corresponding to 300 bp of the 5' end of the human uPAR gene inhibited tumor cell invasion in vitro and tumor formation in vivo. Here we sought to determine whether uPAR is necessary for cell survival and whether the inhibition of tumor formation in nude mice is due to apoptosis of intracerebrally injected SNB19 cells. Apoptosis measured by DNA fragmentation were higher in the brains of animals injected with the antisense stable transfectants than in those injected with the parental cells. Moreover, the increase in apoptotic cell death in vitro was associated with increased expression of apoptotic protein BAX in antisense clones compared to controls. To our knowledge, this is the first report of uPAR playing a novel role in cell survival in human gliomas.

A case of papillary meningioma with a t(1;4)(q44;q21).

We report the results of cytogenetic analyses of three cases of meningiomas. The first case, a papillary meningioma, showed only one cytogenetic abnormality, 46,XX,t(1;4)(q44;q21). In contrast, the other two benign fibroblastic meningiomas showed loss of chromosome 22. Loss and/or rearrangement of chromosomes other than chromosome 22 appears to be associated with a more aggressive clinical course. It is suggested that a sole cytogenetic abnormality with a normal chromosome 22 indicates an atypical nature of meningioma.

Neuroendoscopic approach to tectal tumors: a consecutive series.

OBJECT: The authors report a consecutive series of 10 patients who presented with signs and symptoms caused by tectal tumors. Clinical findings, radiographic features, neuroendoscopic management strategies, and histological findings are reported and discussed. METHODS: Since January 1990, 11 neuroendoscopic procedures were performed in 10 patients who harbored tectal tumors. The patients were followed for an average of 5 years (range 2 months-11 years), and a retrospective study was conducted in which case notes, radiological findings, operative notes, and histopathological findings were assessed. Magnetic resonance (MR) imaging was performed, and the images were used to classify patients into three groups: those with hypertrophy of the tectum in whom isointensity appeared on T1-weighted images (Group 1); those with a tectal tumor occupying the cerebral aqueduct in whom decreased signal intensity appeared on T1-weighted images, as well as no enhancement after gadolinium administration (Group 2); and those with a tectal tumor in whom mixed signal intensity and conspicuous evidence of contrast enhancement appeared on T1-weighted images (Group 3). The results of histological examination were consistent with MR imaging features: in Group 1, glial tissue or gliosis; in Group 2, benign astrocytoma; and in Group 3, malignant astrocytoma. Cerebrospinal fluid diversion was the only surgical treatment that provided relief from obstructive hydrocephalus. One patient in Group 3 underwent radiotherapy and subsequent partial tumor removal under neuroendoscopic guidance. Thereafter, the tumor remained in decline. All patients had normal intellectual status after undergoing surgery in which a neuroendoscope was used. CONCLUSIONS: Neuroendoscopic procedures can provide histological diagnosis, define the tumor-midbrain interrelationship, and be highly effective in treating obstructive hydrocephalus and in removing tectal tumors. This procedure may receive clinical application as a new management strategy for tectal glioma.

Experience with an ultrasonic aspirator in neuroendoscopy.

This report describes the clinical application of an ultrasonic aspirator for endoscopic neurosurgery. Eight patients with intraventricular hematoma with complete tamponade of the ventricular system and marked hydrocephalus were treated with the ultrasonic aspirator for endoscopic neurosurgery. Clinical evaluation and neuroimaging studies were obtained for one month. Removal of hematoma was confirmed on follow-up imaging. The ultrasonic aspirator is operated by electrostriction transducer on low generating power (15-30 watts). Its tube has an outer diameter of 1.8 mm. This aspirator is designed to fragment and aspirate the cattle-liver. On observing the adjacent neural and vascular structures with the neuroendoscope, the massive hematoma within the ventricles is removed with the use of this aspirator. Subtotal removal (> 90% of hematoma volume) was achieved in all patients. All patients tolerated the procedure well. However, postoperative re-rupture occurred in two patients prior to aneurysm clipping following an immediate interval. Three patients with hypertension, two patients with head trauma, and one patient after aneurysm clipping were discharged home within one month after treatment. Preliminary experience with this aspirator has demonstrated its feasibility and safety. Clinical application of this technique is expected in other fields of neurosurgery.

Elevated levels of urokinase-type plasminogen activator and its receptor during tumor growth in vivo.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are important in the regulation of tumor tissue progenesis, cell differentiation, tumor cell motility, and tumor cell invasiveness. We have recently reported that the levels of uPA and uPAR were higher in malignant astrocytomas than in low-grade gliomas. In the present study, we measured the levels of uPA and uPAR during the growth of glioblastomas in nude mice. Using fibrin zymography, densitometry, and an enzyme-linked immunosorbent assay, we found that the enzyme activity and content of uPA were increased 4- to 10-fold during tumor formation. Using a receptor assay and an enzyme linked immunosorbent assay, we found the numbers and content of uPAR were increased 5- to 15-fold during tumor formation. In addition, immunohistochemical staining for uPA and uPAR revealed strong immunoreactivity in tumor cells with the staining more intense on day 28 than on day 14. These results suggest that the upregulation of uPA and uPAR plays a major role in the formation of gliomas.

Neuroendoscopic approach to tectal tumors: a consecutive series.

The authors report a consecutive series of 10 patients who presented with signs and symptoms caused by tectal tumors. Clinical findings, radiographic features, neuroendoscopic management strategies, and histological findings are reported and discussed. Since January 1990, 11 neuroendoscopic procedures were performed in 10 patients who harbored tectal tumors. The patients were followed for an average of 5 years (range 2 months to 12 years), and a retrospective study was conducted in which case notes, radiological findings, operative notes, and histopathological findings were assessed. Magnetic resonance (MR) imaging was performed, and the images were used to classify patients into three groups: those with hypertrophy of the tectum in whom isointensity appeared on T1-weighted images (Group 1); those with a tectal tumor occupying the cerebral aqueduct in whom decreased signal intensity appeared on T1-weighted images, as well as no enhancement after gadolinium administration (Group 2); and those with a tectal tumor in whom mixed signal intensity appeared on T1-weighted images and conspicuous evidence of contrast enhancement (Group 3). The results of histological examination were consistent with MR imaging features: in Group 1, glial tissue or gliosis; in Group 2, benign astrocytoma; and in Group 3, malignant astrocytoma. Cerebrospinal fluid diversion was the only surgical treatment that provided relief from obstructive hydrocephalus. One patient in Group 3 underwent radiotherapy and subsequent partial tumor removal under neuroendoscopic guidance. Thereafter, the tumor remained in decline. All patients had normal intellectual status after undergoing surgery in which a neuroendoscope was used. Neuroendoscopic procedures can provide histological diagnosis, define the tumor-midbrain interrelationship, and be highly effective in treating obstructive hydrocephalus and in removing tectal tumors. This procedure may receive clinical application as a new management strategy for tectal glioma.

Elevated levels of Mr 92,000 type IV collagenase during tumor growth in vivo.

Our previous studies demonstrated that matrix metalloproteinase (MMP-9) levels were significantly higher in human glioblastoma tissue samples than in low-grade brain tumors and normal brain tissue (Rao et al., Cancer Res. 53, 2208-2211, 1993). In the present study, we measured the levels of MMP-2 and MMP-9 during the growth of glial tumors in nude mice by intracerebral injection of glioblastoma cells. Using gelatin zymography, densitometry, and an enzyme-linked immunosorbent assay, we found that the enzyme activity and protein count of MMP-2 and MMP-9 were a respective 3- to 10- and 2- to 30-fold higher in tumors at day 14 and 28 than in normal tissue. Immunohistochemical staining for MMP-9 showed strong immunoreactivity in tumor cells and the staining intensity was much higher at day 28, compared to day 14. These results suggest that upregulation of MMP-9 plays a major role in the glioma tumor growth in vivo.

Effect of cisplatin and BCNU on MMP-2 levels in human glioblastoma cell lines in vitro.

Matrix metalloproteinases (MMPs) play an important role in various physiological and pathological conditions such as tissue remodeling, and cancer cell invasion and metastasis. The aim of this study was to determine the effect of the antitumor compounds cis-dichlorodiammine platinum (ii) (cisplatin) and 1, 3 bis (2-chloroethyl)-1-nitrosourea (BCNU) on 72-kDa type IV collagenase activity (MMP-2) in human gliomas. Human glioblastoma cell lines were treated with cisplatin (25 microM), and BCNU (50 microM), and the levels of MMP-2 were estimated in serum-free conditioned medium and in cell extracts at different time intervals. Gelatin zymography revealed increased levels of MMP-2 in serum-free conditioned medium and in cell extracts of untreated glioblastoma cell cultures during a 72-h period. In contrast, MMP-2 levels were significantly decreased in cisplatin-treated cells both in conditioned medium and cell extracts. However, no significant changes of MMP-2 levels were noted in BCNU-treated cells. Quantitative analysis of MMP-2 enzyme activity by densitometry and amount of MMP-2 protein by ELISA showed significantly decreased levels of MMP-2 in cisplatin-treated cells compared to BCNU and untreated glioblastoma cells. The results indicate that decreased levels of MMP-2 might represent an additional mechanism by which cisplatin provides its antineoplastic effects.

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors.

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5' end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.

Cisplatin but not BCNU inhibits urokinase-type plasminogen activator levels in human glioblastoma cell lines in vitro.

Glioblastomas extensively invade the surrounding normal brain tissue, with a concomitant expression of various proteolytic enzymes, in particular urokinase-type plasminogen activator (uPA). In this study we used cis-diamminedichloroplatinum (cisplatin) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), commonly used anti-cancer drugs for the treatment of glioblastomas, to study the expression of uPA in three human glioblastoma cell lines in vitro. Cells were treated with 25 microM cisplatin and 50 microM BCNU, and uPA levels were estimated by fibrin zymography during a 72-h time course. Treatment of glioblastoma cells with cisplatin resulted in significantly decreased levels of uPA in serum-free conditioned medium and cell extracts, compared to BCNU-treated and untreated cell lines. Quantitative levels of uPA enzyme activity assessed by scanning laser densitometry and uPA protein by ELISA using antibody against uPA showed decreased levels of uPA in cisplatin-treated glioma cell lines relative to BCNU and untreated cell lines. Our results suggest that anti-tumor compound, cisplatin, may exert its anti-neoplastic effects by inhibiting uPA in malignant glioblastomas.

In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor.

The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Significant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were significantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any significant difference however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.

Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and Gbeta/gamma-dependent signaling pathways.

Shear stress differentially regulates production of many vasoactive factors at the level of gene expression in endothelial cells that may be mediated by mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and N-terminal Jun kinase (JNK). Here we show, using bovine aortic endothelial cells (BAEC), that shear stress differentially regulates ERK and JNK by mechanisms involving Gi2 and pertussis toxin (PTx)-insensitive G-protein-dependent pathways, respectively. Shear activated ERK with a rapid, biphasic time course (maximum by 5 min and basal by 30-min shear exposure) and force dependence (minimum and maximum at 1 and 10 dyn/cm2 shear stress, respectively). PTx treatment prevented shear-dependent activation of ERK1/2, consistent with a Gi-dependent mechanism. In contrast, JNK activity was maximally turned on by a threshold level of shear force (0.5 dyn/cm2 or higher) with a much slower and prolonged time course (requiring at least 30 min to 4 h) than that of ERK. Also, PTx had no effect on shear-dependent activation of JNK. To further define the shear-sensitive ERK and JNK pathways, vectors expressing hemagglutinin epitope-tagged ERK (HA-ERK) or HA-JNK were co-transfected with other vectors by using adenovirus-polylysine in BAEC. Expression of the mutant (alpha)i2(G203), antisense G(alpha)i2 and a dominant negative Ras (N17Ras) prevented shear-dependent activation of HA-ERK, while that of (alpha)i2(G204) and antisense (alpha)i3 did not. Expression of a Gbeta/gamma scavenger, the carboxyl terminus of beta-adrenergic receptor kinase (betaARK-ct), and N17Ras inhibited shear-dependent activation of HA-JNK. Treatment of BAEC with genistein prevented shear-dependent activation of ERK and JNK, indicating the essential role of tyrosine kinase(s) in both ERK and JNK pathways. These results provide evidence that 1) Gi2-protein, Ras, and tyrosine kinase(s) are upstream regulators of shear-dependent activation of ERK and 2) that shear-dependent activation of JNK is regulated by mechanisms involving Gbeta/gamma, Ras, and tyrosine kinase(s).

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.