Use of monopolar cautery in patient with a vagal nerve stimulator during neuromuscular scoliosis surgery.

It is a historic and common practice while performing spine surgery on patients with a VNS has been to have the patient's neurologist turn off the VNS generator in the pre-operative anesthetic care unit and to use bipolar rather than monopolar electrocautery. Here we report a case of a 16-year-old male patient with cerebral palsy and refractory epilepsy managed with an implanted VNS who had scoliosis surgery (and subsequent hip surgery) conducted with the use of monopolar cautery. Although VNS manufacturer guidelines suggest that monopolar cautery should be avoided, perioperative care providers should consider its selective use in high-risk instances (with greater risks of morbidity and mortality due to blood loss which outweigh the risk of surgical re-insertion of a VNS) such as cardiac or major orthopedic surgery. Considering the number of patients with VNS devices presenting for major orthopedic surgery is increasing, it is important to have an approach and strategy for perioperative management of VNS devices.

The role of surgery in the management of intracranial gliomas: current concepts.

The role of surgery in the management of human gliomas has been controversial. The results from numerous neurosurgical series are inconsistent. The current adjuvant therapies have facilitated treatment of patients, and have rendered neurosurgical removal without morbidity or mortality more commonplace than ever before. Here, we investigated the role of surgery in the management of adults with low- and high-grade gliomas. Even though there is substantial evidence which claims that surgery per se has a role to play in extending patient survival, there is a paucity of randomized clinical trials on this subject, and little in the way of Class II data to support these claims. However, this should not divert patients away from surgery, because there may be additional benefits from a concerted effort to remove a tumor completely. At the present time, it seems best that clinicians continue to individualize patient treatment based on a myriad of factors that relate to the patient, the patient's tumor, and the known biology of the disease.

Magnetoencephalography for surgical treatment of refractory status epilepticus.

Magnetoencephalography (MEG) provides accurate localizing information of the epileptogenic zones in localization-related epilepsies. Refractory status epilepticus (RSE) is a life-threatening emergency that often requires prolonged high-dose suppressive therapy (HDST) to stop frequent and prolonged seizures. Surgical treatments for patients with RSE secondary to pre-existing epilepsy were reported. This article addresses the role of MEG in localizing the epileptogenic zone for the surgical treatment of patients with RSE. Five pediatric patients with RSE underwent epilepsy surgery using MEG, scalp video EEG and magnetic resonance imaging (MRI). Ictal MEG spike sources (MEGSSs) were localized in the clustered interictal MEGSSs in right Rolandic region (patient 3) and right temporal region (patient 5). Interictal MEG revealed unilateral clustered MEGSSs in four patients (patients 1, 2, 4, and 5) and bilateral (patient 3). Ictal-onset EEG findings were localized to one region in three patients (patients 1, 3, and 5) and two regions in the other two patients (patients 2 and 4). In all five patients, interictal discharges were widespread involving over two lobes (patients 2 and 4) or three lobes (patients 1, 3, and 5). Suppression burst pattern was obtained by HDST (patient 5). MRI showed cortical dysplasia in three patients (patients 1, 3, and 4). Patient 2 had a normal MRI. Patient 5 had normal MRI at the onset. Repeat MRI 5 days later showed diffusion restriction in the right hippocampus associated with increased signal intensity on T2 and FLAIR sequences. We performed cortical excision in two patients (patients 1 and 4), hemispherectotomy one (patient 3) and anterior temporal lobectomy two patients (patients 2 and 5). Two patients (patients 1 and 3) became seizure free, the other three patients experienced residual seizures. MEG showed clustered MEGSSs during the RSE in the pre-existing epilepsy patients and at an early time window in the acute symptomatic RSE patients. The complete resection of clustered MEGSSs can control RSE and possibly lead to a seizure free outcome.

Magnetoencephalography for surgical treatment of refractory status epilepticus.

Magnetoencephalography (MEG) provides accurate localizing information of the epileptogenic zones in localization-related epilepsies. Refractory status epilepticus (RSE) is a life-threatening emergency that often requires prolonged high-dose suppressive therapy (HDST) to stop frequent and prolonged seizures. Surgical treatments for patients with RSE secondary to pre-existing epilepsy were reported. This article addresses the role of MEG in localizing the epileptogenic zone for the surgical treatment of patients with RSE. Five pediatric patients with RSE underwent epilepsy surgery using MEG, scalp video EEG and magnetic resonance imaging (MRI). Ictal MEG spike sources (MEGSSs) were localized in the clustered interictal MEGSSs in right Rolandic region (patient 3) and right temporal region (patient 5). Interictal MEG revealed unilateral clustered MEGSSs in four patients (patients 1, 2, 4, and 5) and bilateral (patient 3). Ictal-onset EEG findings were localized to one region in three patients (patients 1, 3, and 5) and two regions in the other two patients (patients 2 and 4). In all five patients, interictal discharges were widespread involving over two lobes (patients 2 and 4) or three lobes (patients 1, 3, and 5). Suppression burst pattern was obtained by HDST (patient 5). MRI showed cortical dysplasia in three patients (patients 1, 3, and 4). Patient 2 had a normal MRI. Patient 5 had normal MRI at the onset. Repeat MRI 5 days later showed diffusion restriction in the right hippocampus associated with increased signal intensity on T2 and FLAIR sequences. We performed cortical excision in two patients (patients 1 and 4), hemispherectotomy one (patient 3) and anterior temporal lobectomy two patients (patients 2 and 5). Two patients (patients 1 and 3) became seizure free, the other three patients experienced residual seizures. MEG showed clustered MEGSSs during the RSE in the pre-existing epilepsy patients and at an early time window in the acute symptomatic RSE patients. The complete resection of clustered MEGSSs can control RSE and possibly lead to a seizure free outcome.

Magnetoencephalographic spike sources associated with auditory auras in paediatric localisation-related epilepsy.

OBJECTIVE: To characterise magnetoencephalographic spike sources in paediatric patients with auditory auras and recurrent localisation-related epilepsy. METHODS: Six patients (four boys and two girls (ages 7-14 years) were retrospectively studied. All patients had auditory auras as part of their initial seizure manifestation, including four patients who underwent previous brain surgery. Scalp video electroencephalography and magnetoencephalography (MEG) were carried out in six patients, intraoperative electrocorticography in three patients and extraoperative intracranial video electroencephalography in one patient. MEG auditory-evoked fields (AEFs) were studied in four patients. RESULTS: Three patients had elementary auditory auras, one had complex auditory aura and two had both complex and elementary auras. All six patients had clustered MEG spike sources with coexisting scattered spike sources. MEG clusters were localised in the superior temporal gyrus with surrounding scatters in four patients (two left and two right); two patients had scattered spikes in the superior temporal gyrus in addition to clustered MEG spike sources in the left inferior and middle frontal gyri or parieto-occipital region. AEFs were located within an MEG cluster in one patient and within 3 cm of a cluster in two patients. Surgical resection, including the regions of MEG clusters, was carried out in four patients. Three of four patients who had previous surgeries were seizure free at 2 years after excision of the MEG cluster region. CONCLUSIONS: MEG spike sources clustered in the superior temporal gyrus in six patients with auditory auras. These spike sources were in close proximity or seemed to engulf the magnetic AEF. Areas with MEG spike sources contained the residual or recurrent epileptogenic zone after incomplete cortical excision for lesional epilepsy.

Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity.

Glial fibrillary acidic protein (GFAP) is a M(r) 50,000 intracytoplasmic filamentous protein that constitutes a portion of, and is specific for the cytoskeleton of the astrocyte. GFAP appears to stabilize the astrocyte's cytoskeleton, and helps maintain normal astrocyte cell shape through complex interactions between it and the nuclear and plasma membranes. A critical observation from histopathological studies is that there is progressive loss of GFAP expression with increasing astrocytic anaplasia. To determine how GFAP expression affects human malignant astrocytoma cells that are GFAP-deficient, we have stably transfected GFAP-negative SF-126 human astrocytoma cells with a eukaryotic expression vector into which a complementary DNA for the entire coding sequence of the human GFAP protein has been inserted. Positive transformants were selected for neomycin resistance, and GFAP-positive astrocytoma cells have been identified by indirect immunofluorescence using anti-GFAP antibodies. Upon successful transfection, 5 stably transfected SF-126 astrocytoma cell clones were selected that produced GFAP, formed elongated processes in cell culture, and demonstrated decreased proliferation in a tritiated thymidine uptake assay when compared to the control. In addition, the 5 astrocytoma cell clones demonstrated marked reduction in the number and growth of colonies in soft agar when compared to the control. Southern analysis revealed extra copies of the GFAP complementary DNA that had become integrated into the SF-126 astrocytoma cell genome. Northern analysis revealed variable expression of a 3.5-kilobase GFAP mRNA transcript among the 5 cell clones. The amount of GFAP mRNA expression and immunoreactivity by the stably transfected SF-126 astrocytoma cell clones was found to correlate inversely with astrocytoma proliferation and growth in soft agar. The restoration of GFAP expression to GFAP-negative human astrocytoma cells has affected tumor cell morphology, proliferation, and growth in soft agar in this experimental cell system.

Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells.

Glial fibrillary acidic protein (GFAP)-positive astrocytoma cells were stably transfected with an expression vector carrying a murine complementary DNA for GFAP in the antisense orientation. Three stably transfected GFAP-negative transformants were identified by indirect immunofluorescence and expanded in vitro. The stably transfected and control cell clones were analyzed for morphological alterations, growth in monolayer and soft agar, adhesiveness, and in vitro invasive potential. In contrast to control astrocytoma cells which retained an astrocytic phenotype with polygonal or triangular cells extending multiple long and thin processes, the antisense GFAP-transfected cells demonstrated marked morphological alterations in the form of flat, epithelioid cells devoid of long, astrocytic glial processes. The antisense GFAP-transfected clones demonstrated a greater degree of cell crowding and piling at confluence than did controls. By tritiated thymidine analysis, the antisense GFAP-transfected cell clones demonstrated a 2-3-fold increase in incorporation of the radiolabel, suggesting an enhanced proliferative potential over controls. Antisense GFAP-transfected astrocytoma clones formed larger and more numerous colonies than did controls when tested for anchorage-independent growth in soft agar. Following a time-course adhesion assay, antisense GFAP-transfected astrocytoma clones were found to be less adherent to their substratum than controls. When assessed in an in vitro invasion assay system, antisense GFAP-transfected astrocytoma cells more readily penetrated Matrigel-coated filters than did controls. These data have shown that eliminating GFAP expression from astrocytoma cells has affected astrocytoma cell morphology and adhesion. The data also suggest that the growth and invasive potential of the antisense GFAP-transfected astrocytoma cells have been significantly enhanced by altering the expression of this glial-specific cytoskeletal protein in this experimental cell system.

Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins.

We devised a model system to study the effects of extracellular matrix proteins on the malignant phenotype of an anaplastic glioma cell line, U 343 MG-A. Well-characterized cultures derived from normal human leptomeninges were grown to confluence and maintained for 2 weeks. The leptomeningeal cells were then removed with base and detergent, leaving behind an extracellular matrix enriched in laminin, fibronectin, type I and IV collagen, and procollagen III. U 343 MG-A tumor cells planted on top of this normal extracellular matrix were profoundly growth inhibited compared with glioma cells grown on plastic alone. Glioma cells grown on the extracellular matrix developed multiple, slender processes and assumed a more differentiated astrocytic phenotype; immunostains for glial fibrillary acidic protein revealed a more extensive intracytoplasmic network of intensely staining filaments than in control glioma cells. When glioma cells grown on the extracellular matrix were analyzed by an enzyme-linked immunosorbent assay for glial fibrillary acidic protein, the amount of this intermediate filament per cell was increased 20-fold compared with glioma cells growing on plastic. The growth and differentiation of U 343 MG-A glioma cells in flasks coated with purified fibronectin or laminin was not significantly perturbed; however, glioma cell cultures grown in flasks coated with purified type I or IV collagen showed decreased cellular proliferation, stellate cell formation, and increased levels of glial fibrillary acidic protein per cell compared with glioma cells growing on plastic. Gelatin gel analysis showed that U 343 MG-A glioma cells growing on plastic secreted a 65,000-D metalloproteinase that was not secreted by glioma cells grown on the leptomeningeal extracellular matrix. We conclude that in this system, the extracellular matrix of a normal human leptomeningeal culture substantially inhibited the proliferation of and induced differentiation in an anaplastic glioma cell line. Our analysis of single components of the extracellular matrix suggests that these effects may be mediated in part by type I and IV collagen. The mechanism by which the leptomeningeal extracellular matrix inhibits glioma cell proliferation may be by diminishing tumor-associated protease secretion so that the degradation of extracellular matrix macromolecules in the tumor cell microenvironment is prevented and tumor cell migration becomes less likely.

Establishment and characterization of a cell line from a human gliosarcoma.

A human gliosarcoma culture was characterized from the time of inception to the time of establishment of the cell line (SF-539 BT). Immunohistochemical analysis of the original tumor showed 2 distinct regions of cells. The gliomatous regions were identified by immunostains for glial fibrillary acidic protein and the sarcomatous regions by immunostains for laminin, collagen type IV, procollagen type III, and fibronectin. In early-passage culture, both types of cells maintained their characteristic immunohistochemical profiles; however, after the fourth subcultivation in monolayer culture, no cells expressing glial fibrillary acidic protein could be identified. All cells had become morphologically uniform and expressed laminin, collagen type IV, procollagen type III, and fibronectin only. The immunostaining profile of clones grown in soft agar was similar to that of cells in monolayer culture. At establishment, SF-539 BT has a saturation density of 1.3 X 10(6) cells/25 sq cm, a doubling time of 32 h, and a plating efficiency of 22% in monolayer culture. The tumor cell line is resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, has an abnormal karyotype, grows anchorage independently, and forms a tumor that most closely resembles a spindle cell sarcoma in athymic mice. Its ultrastructure in monolayer culture consists of large cells with an expanded rough endoplasmic reticulum and abundant multivesicular bodies; in athymic mice, extracellular collagen fiber formation is prominent. DEAE-cellulose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cultures labeled with [3H] proline demonstrated interstitial collagen formation. We conclude that the cell line at establishment is a collagen-producing spindle cell sarcoma that resembles the sarcomatous regions of the original mixed tumor. Further cell separation and characterization studies are needed to determine the pathogenesis of mixed tumors such as gliosarcoma.

Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells.

Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.

Retinoic acid and the cyclin dependent kinase inhibitors synergistically alter proliferation and morphology of U343 astrocytoma cells.

We have characterized the expression and activity of the cell cycle regulatory machinery and the organization of the cytoskeleton of the p16(Ink4a)-deficient astrocytoma cell line, U343 MG-a (U343), following retinoic acid (RA) treatment. RA causes cell cycle arrest at low cell density and significant morphological changes in U343 cells, reflected by reorganization of the intermediate filament, GFAP, and actin. RA-induced cell cycle arrest is also associated with induction of p27Kip1 expression, inhibition of cdk2-associated kinase activity and alteration of the phosphorylation state of the pRB-family proteins. We next determined the effect of inducing expression of the cyclin dependent kinase inhibitors (CKI's), p16(Ink4a), p21Cip1/Waf1 or p27Kip1 on the proliferation and morphology of these malignant astrocytoma cells in the absence and presence of RA. Induction of p16, p21 or p27, using the tetracycline repressor system, potently inhibits proliferation of U343 cells. However, rather than resembling RA-treated cells, CKI-induced U343 cells become flat with abundant cytoplasm and perinuclear vacuolization. CKI-induced morphological alterations are accompanied by a significant reorganization of glial filaments within the cytoplasm. Interestingly, when U343 cells are growth arrested by p16, p21 or p27 induction and treated simultaneously with RA, a dramatic morphological change occurs, cells acquiring multiple long, tapering processes reminiscent of primary astrocytes. This rearrangement is accompanied by reorganization of GFAP, vimentin and actin. Vimentin specifically relocalizes to the tips of the long processes which form. The arrangement of intermediate filaments in these cells is, in fact, indistinguishable from their arrangement in primary human astrocytes. These data demonstrate that when a strong proliferative block, produced by CKI expression, occurs in conjunction with the morphogenic signals generated by RA, these p16-deficient malignant astrocytoma cells are induced to phenotypically resemble normal astrocytes.

The E2F-family proteins induce distinct cell cycle regulatory factors in p16-arrested, U343 astrocytoma cells.

We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, deltap34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2F's altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2F's uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.

Epilepsy surgery: recent advances in brain mapping, neuroimaging and surgical procedures.

The general principle of epilepsy surgery is to achieve seizure freedom without causing any neurological deficit that would outweigh the clinical benefit. To achieve this, the epileptogenic zone, which is the part of the brain responsible for seizure generation, as well as the anatomic location of the eloquent cortex must be precisely identified in order to spare those functions during excision of the epileptogenic tissue. Major technical advances over the last decade have continuously contributed to increase our ability to map the brain and identify these critical areas. These technologies and innovations that can be routinely used today include non-invasive studies such as magnetoencephalography (MEG), functional MRI (fMRI), simultaneous EEG-fMRI, and nuclear medicine based methods like PET and SPECT as well as invasive studies through chronically implanted electrodes. Electrodes can be either placed subdurally via burr holes and craniotomies or within the brain parenchima via frame-based and frameless stereotactic methods. Apart from a continuous change in these insertion techniques, the most valuable advances here include recordings on high frequency bandwidth (100-600 Hz EEG) that are capable to delineate high-frequency oscillations (HFOs). These HFOs have been recognized as a biomarker for epileptogenic tissue. All of these technical advances have made epilepsy surgery a truly multidisciplinary field and surgeons have to be able to understand and interpret all of the gathered data. Moreover, this development has influenced surgical approaches and techniques and epilepsy surgery today includes a wide variety of procedures. These can be subdivided into resective, disconnective and neuromodulation procedures and vary from a small, targeted lesionectomy to disconnection/resection of one entire hemisphere. This review will give an overview of the available surgical techniques today and will focus on how the technical advances enable us to map the brain and delineate the critical areas.

Characterization of glial filament-cytoskeletal interactions in human astrocytomas: an immuno-ultrastructural analysis.

The role that glial filaments play in cells and tumors of glial origin is not well understood. We therefore undertook the present study to determine the relationships between glial and vimentin intermediate filaments (IFs), actin microfilaments, and CD44, a cell surface glycoprotein important in cell migration and invasion, in human astrocytoma cells. Three astrocytoma cell lines, U343 MG-A (U343), U251 MG (U251), and antisense GFAP-transfected U251 (asU251) were studied using immunofluorescence confocal and immunoelectron microscopy. Furthermore, we studied the phenotypic behaviour of these astrocytoma cell lines by analyzing their migration through Matrigel in vitro. U343 astrocytoma cells had the highest expression levels of glial fibrillary acidic protein (GFAP), whereas asU251 had virtually no expression of GFAP. Parental U251 cells had intermediate expression levels of GFAP. The elimination of GFAP expression in as U251 cells was accompanied by a marked increase in vimentin, actin microfilaments and CD44 levels. Gold labeling density counts of cytoskeletal and cell surface elements demonstrated that the differences between GFAP, actin, CD44 and vimentin levels in the different astrocytoma cell lines were statistically significant (p < 0.05). Results from the in vitro invasion assay revealed that U343 cells demonstrated the least invasive potential, whereas asU251 astrocytoma cells demonstrated the most. Our results show that elimination of GFAP expression by antisense leads to marked alterations in cell morphology and phenotypic behaviour. These data imply that GFAP may be linked spatially and functionally to cytoskeletal elements which may be altered when this IF is deleted in astrocytomas.

Tropoelastin and elastin degradation products promote proliferation of human astrocytoma cell lines.

Expression of tropoelastin, the precursor of insoluble elastin and a major component of elastic fibers, has not yet been demonstrated in astrocytomas nor has it been linked to their proliferation. Here we report that human astrocytoma cell lines (U87 MG, U251 MG, U343 MG-A, U373 MG, SF 126, SF188, SF 539), as well as surgical specimens of malignant human astrocytomas, express intracellular tropoelastin. The tropoelastin produced by astrocytoma cells is, however, susceptible to proteolytic trimming to the extent that it cannot be assembled into extracellular elastic fibers. Astrocytoma cells also express the cell surface 67-kDa elastin binding protein (EBP), which binds elastin degradation products, leading to the upregulation of cyclin A and cdk2 and increased incorporation of [3H]-thymidine. The elastin-dependent mitogenic response of astrocytoma cells is abolished by lactose and chondroitin sulfate, factors which cause shedding of this 67-kDa elastin receptor from the cell surface and by blocking anti-EBP antibody. We therefore suggest that, in astrocytomas, endogenous tropoelastin degradation products bind to EBP and generate signals leading to cell cycle progression in an autocrine or paracrine manner. This is the first report implicating elastin-derived peptides as possible mitogens in malignant astrocytomas.

Cyclin and cyclin-dependent kinase expression in human astrocytoma cell lines.

The expression of cyclins (A, B1, D1, D3, E), cyclin-dependent kinases (CDK2(3), CDK4), and the cyclin-dependent kinase inhibitors (CDKIs) p16(INK4A) and p21(CIP1) was studied in 9 malignant human astrocytoma cell lines using northern blot analysis, immunocytochemistry, and immunoblotting to see if their altered expression contributed to astrocytoma proliferation. Steady state cell cycle mRNA expression was analyzed in unsynchronized tumor cells, and cell cycle phase-specific gene expression was analyzed in 3 synchronized cell lines. Analysis of steady state expression revealed increased levels of several different cyclin transcripts and CDKs in a number of astrocytoma cell lines compared with normal human brain tissue or cultured fibroblasts. We confirm previous reports identifying loss of p16(INK4A) in astrocytomas, as a p16(INK4A) transcript was identified in only 2 cell lines and protein in 1 cell line. However, we now show that p21(CIP1) expression was also diminished relative to normal fibroblasts in all astrocytoma cell lines studied regardless of p53 mutation status. Analysis of synchronized astrocytoma cells revealed altered timing of mRNA expression of several cyclins. Immunocytochemistry revealed a generalized increase in immunoreactivity of astrocytoma cells for most cyclins and CDKs compared with human fibroblasts. Immunoblotting also revealed increased expression of cyclin proteins in a number of astrocytoma cell lines. These data suggest that increased expression of cyclins and CDKs, and decreased expression of CDKIs by human astrocytoma cell lines may contribute to their increased proliferative state. In addition, our data show that alterations in cell cycle genes in astrocytomas are not confined to the cyclin D1-CDK4-pRb axis.

Transfection of an invasive human astrocytoma cell line with a TIMP-1 cDNA: modulation of astrocytoma invasive potential.

Malignant astrocytomas are highly invasive tumors which infiltrate diffusely into regions of normal brain. The degradation of the extracellular matrix (ECM) by matrix metalloproteinases is thought to be one of the most important steps in the process of tumor invasion. However, the activity of most matrix metalloproteinases (MMPs) can be modulated by simultaneously secreted inhibitors (tissue inhibitors of metalloproteinases, TIMPs). We have previously shown that an imbalance between the levels of MMPs and TIMPs may be essential in the determination of the invasiveness of certain human malignant astrocytoma cell lines. To determine if the up-regulation of TIMP genes and gene products could modulate the invasiveness of human malignant astrocytoma cells, in the present study we have transfected a highly invasive astrocytoma cell line, SF-188, with an expression vector carrying a full-length TIMP-1 cDNA. The parental SF-188 astrocytoma cell line overexpresses the 72-kDa and 92-kDa type IV collagenases with little expression of TIMPs-1 and -2. Following transfection with TIMP-1, SF-188 astrocytoma clones expressed the 0.9 kb TIMP-1 message by northern analysis, and produced a 21 kDa metalloproteinase inhibitor by reverse zymography. The stable TIMP-1 SF-188 transformants demonstrated morphological changes and diminished growth rates in soft agar when compared to controls. The invasion of successfully TIMP-1 transfected astrocytoma cells across matrigel-coated filters was significantly decreased over controls. These results suggest that upregulation of TIMP-1 expression in SF-188 astrocytoma cells has decreased their in vitro invasive potential.

An ultrastructural and immunocytochemical analysis of leptomeningeal and meningioma cultures.

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression.

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.