Shin Jung

Hyaluronate receptors mediating glioma cell migration and proliferation.

The extracellular matrix (ECM) of the central nervous system (CNS) is enriched in hyaluronate (HA). Ubiquitous receptors for HA are CD44 and the Receptor for HA-Mediated Motility known as RHAMM. In the present study, we have investigated the potential role of CD44 and RHAMM in the migration and proliferation of human astrocytoma cells. HA-receptor expression in brain tumor cell lines and surgical specimens was determined by immunocytochemistry and western blot analyses. The ability of RHAMM to bind ligand was determined through cetylpyridinium chloride (CPC) precipitations of brain tumor lysates in HA-binding assays. The effects of HA, CD44 blocking antibodies, and RHAMM soluble peptide on astrocytoma cell growth and migration was determined using MTT and migration assays. Our results show that the expression of the HA-receptors, CD44, and RHAMM, is virtually ubiquitous amongst glioma cell lines, and glioma tumor specimens. There was a gradient of expression amongst gliomas with high grade gliomas expressing more RHAMM and CD44 than did lower grade lesions or did normal human astrocytes or non-neoplastic specimens of human brain. Specific RHAMM variants of 85- and 58-kDa size were shown to bind avidly to HA following CPC precipitations. RHAMM soluble peptide inhibited glioma cell line proliferation in a dose-dependent fashion. Finally, while anti-CD44 antibodies did not inhibit the migration of human glioma cells, soluble peptides directed at the HA-binding domain of RHAMM inhibited glioma migration both on and off an HA-based ECM. These data support the notion that HA-receptors contribute to brain tumor adhesion, proliferation, and migration, biological features which must be better understood before more effective treatment strategies for these tumors can be found.

Co-expression of nestin and vimentin intermediate filaments in invasive human astrocytoma cells.

Intermediate filaments (IFs) are highly diverse intracytoplasmic proteins within the cytoskeleton which exhibit cell type specificity of expression. A growing body of evidence suggests that IFs may be involved as collaborators in complex cellular processes controlling astrocytoma cell morphology, adhesion and proliferation. As the co‐expression of different IF subtypes has been linked to enhanced motility and invasion in a number of different cancer subtypes, we undertook the present study to examine the expression of vimentin and nestin in a panel of human astrocytoma cell lines whose tumorigenicity, invasiveness and cytoskeletal protein profiles are well known. Astrocytoma cells were examined for IF protein expression by immunofluorescence confocal and immunoelectron microscopy. The motility of all cell lines was determined by computerized time‐lapse videomicroscopy. Invasive potential of astrocytoma cells was determined using Matrigel as a barrier to astrocytoma cell invasion in vitro. Vimentin was expressed by all astrocytoma cell lines. On the other hand, nestin was variably expressed among the different cell lines. The most motile and invasive astrocytoma cell line in our study was antisense GFAP‐transfected U251 (asU251) astrocytoma cells which showed marked up‐regulation of nestin expression compared to the U251 parental cell line and controls. The U87 astrocytoma cell line also demonstrated high nestin expression levels and was associated with an increased basal motility rate and a high degree of invasiveness through Matrigel. U343 astrocytoma cells did not express nestin, but had high levels of GFAP. It had the lowest motility rate and invasiveness of all the astrocytoma cell lines examined. Taken together, these data suggest that for the astrocytoma cell lines examined in this study, nestin and vimentin co‐expression may serve as a marker for an astrocytoma cell type with enhanced motility and invasive potential. Further studies are required to determine the mechanism by which dual‐IF protein expression alters other cytoskeletal or cell surface receptor protein components important in the process of astrocytoma invasion.

Expression of p57KIP2 Potently Blocks the Growth of Human Astrocytomas and Induces Cell Senescence

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.

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 MO, 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.

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.

Astrocytoma cell interaction with elastin substrates: Implications for astrocytoma invasive potential

Elastin has been identified within the meninges and the microvasculature of the normal human brain. However, the role that elastin plays in either facilitating astrocytoma cell attachment to these structures or modulating astrocytoma invasion has not been previously characterized. We have recently shown that astrocytoma cell lines and specimens produce tropoelastin, and express the 67 kDa elastin binding protein (EBP*). In the present report, we have established that astrocytoma cells attach to elastin as a substrate in vitro. The U87 MG astrocytoma cell line demonstrated the greatest degree of adhesion. In addition, all astrocytoma cell lines examined were capable of penetrating and migrating through an intact elastin membrane, and of degrading tritiated‐elastin, a process that could be prevented by the pre‐incubation of astrocytoma cells with EDTA, but not with α1‐antitrypsin. Astrocytoma cells were also capable of penetrating 1 mm sections of human brain tissue maintained as organotypic cultures. Interestingly, the invasive potential of cultured astrocytoma cells plated on organotypic cultures of human brain was significantly increased after exposure to elastin degradation products (κ‐elastin), which interact with astrocytoma cell surface EBP. Our data show that astrocytoma cells express a functional 67 kDa EBP, enabling them to potentially recognize and attach to elastin as a substrate. These data also suggest that this elastin receptor may be involved in processes which regulate regional astrocytoma invasion. GLIA 25:179–189, 1999.

Cell surface aggregation of elastin receptor molecules caused by suramin amplified signals leading to proliferation of human glioma cells

Abstract We have recently shown that glioma cell lines, as well as cells of human malignant gliomas in situ, synthesize tropoelastin. In addition, glioma cells degrade tropoelastin using metalloproteinase(s), and the resulting peptides, incapable of assembling in the extracellular fibers, interact with the 67-kDa cell surface elastin binding protein (EBP), to transduce signals leading to up-regulation of cell proliferation. In this report, we show that exposure to the polysulfonated bis-naphthylurea suramin causes accumulation of physiologically active EBP molecules on the cell surface of a panel of glioma cell lines (U87, MG, U251 MG, U343 MG-A, U373 MG, SF 126, SF188, SF539), which results in an increase of cellular attachment to elastin-coated dishes and in an efficient binding of radiolabeled tropoelastin. Moreover, 100–200 μM suramin stimulates [3H]-thymidine incorporation by those tropoelastin-producing glioma cell lines, but not by A 2058 melanoma cells, which do not produce elastin. Treatment of all glioma cell lines with 100 μM suramin consistently increased expression of cyclin A and its cyclin-dependent kinase, cdk 2, to levels reached following the exposure to exogenous elastin-degradation products (κ-elastin). Our data suggest that a suramin-stimulated accumulation of EBP molecules on the cell surface of glioma cells amplifies the elastin-derived signals, leading to their progression through the cell cycle.

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.

Cell cycle control in pediatric neuro-oncology

Introduction Brain tumors are the most common solid tumor of childhood, second in frequency only to leukemia [1]. Astrocytomas represent the most common primary brain tumor of childhood comprising almost 50% of all brain tumors in this age group [2]. Of these, 20% are high-grade, or anaplastic lesions. Unlike adults with low-grade astrocytomas who frequently progress to higher grade lesions over time, the malignant transformation of a low-grade astrocytoma to highgrade one in a child is a rare event [3]. Medulloblastoma is another common brain tumor in childhood accounting for about 25% of pediatric brain tumors. This tumor is highly proliferative with a propensity to invade the fourth ventricle as well as the overlying subarachnoid spaces. As such, medulloblastoma is characterized by widespread dissemination along cerebrospi-nal fluid pathways. Unfortunately, many children who suffer from malignant astrocytoma and medulloblastoma will die from their tumors despite recent advances in surgery, radiation, and chemotherapy [4]. Improving the prognosis for these children will involve not only the identification of better treatment modalities, but a greater understanding of the pathogenetic basis for these tumors. Recently, several studies have shown that a class of genes which control cell cycle regulation play an important role in the pathogenesis of several different human tumors, including brain tumors. Uncontrolled cellular proliferation is a hallmark of neoplasia. Experimental studies on the mechanisms of cell proliferation have begun to elucidate a basic tenet in cell cycle dysregulation in tumors, namely that uncontrolled proliferation may be caused by altered expression of positive growth regulators such as cyclins and cyclin-dependent kinases (CDKs), or the loss of negative regulators, including CDK inhibitors (CDKIs), and the retino-blastoma protein (pRB). As a result, a cell is no longer able to respond to important internal or external cues that check its growth. In this review, we will discuss the impact of markers of tumor proliferation on our understanding of tumor growth and response to therapy; we will examine the alterations in several cell cycle regulatory genes as they have been described for human brain tumors with an emphasis on those described in pediatric brain tumors; finally, we will provide data which argue cogently for the targeting of cell cycle genes as a novel means by which the process of cell proliferation in pediatric brain tumors can be directly inhibited. Cell Proliferation Markers The human brain is a unique organ from a cell kinetic standpoint. Neurons become incapable of cell division in the early postnatal period. Although glial cells retain their proliferative potential, as is demonstrated in the process of reactive gliosis, it is still unclear what the initiating events are that transform a normal cell into a highly proliferative malignant brain tumor cell. However, a number of studies have now been performed which have characterized the proliferative indices of human brain tumors, including pediatric brain tumors. The original cell kinetic studies conducted by Hoshino et al. [5–9] on brain tumors

Recent Developments in the Molecular Biology of Human Brain Tumors

Many of the most important scientific achievements within the field of medicine have come to light within the past 40 years. Watson and Crick discovered the structure of DNA in 1952. The genetic code of the human genome was described by 1965. The utility of restriction endonucleases- enzymes that cut large pieces of DNA into smaller, more manageable fragments-was recognized in the 1970s. Gene transfer techniques were described for the first time in 1972. The polymerase chain reaction (PCR) was described in 1985. Tumor suppressor genes, the guardians of the human genome against neoplastic change, were described in 1987. By 1995, more than 1000 patients were treated with gene therapy protocols for a variety of human diseases, including human brain tumors. The recently cloned sheep from chromosomal material contained within fully differentiated sheep mammary epithelial cellsif1ustrates the amazing strides that have occurred since the advent of these molecular biology techniques. This review presents some of these recent advances in the basic science of human brain tumors, specifically in brain tumor invasion, apoptosis, cell cycle, tumor suppressor genes, and signal transduction.