Vitronectin, a glioma-derived extracellular matrix protein, protects tumor cells from apoptotic death.

Vitronectin (VN) is an extracellular matrix (ECM) protein, the synthesis of which in vivo by glioma cells correlates with tumor grade. Although the role of VN as a permissive substrate for glioma migration has been well characterized, its role in conferring a survival advantage for tumor cells has not been addressed previously. By using an in vitro assay of DNA fragmentation as a quantitative measure of apoptotic cell death, we sought to determine whether the sensitivity of two human glioma cell lines (D54 and U251) to drug-induced apoptosis could be inhibited by VN. As well, the extent to which apoptosis could be inhibited was correlated with the levels of the Bcl-2 family of proteins that are known to modulate apoptosis and chemoresistance. Results of the study were: (a) VN coatings, in a dose-dependent manner, inhibited topoisomerase (Topo)-induced apoptosis by up to 50% (optimal coating density, 500 ng/cm2); in contrast, fibronectin (FN), an ECM protein present in abundance in the brain, demonstrated no protection; (b) in a dose-response study, VN clearly conferred a survival advantage (LD50 of Topo: on VN, 120 ng/ml; on FN, 35 ng/ml); (c) the protective effect of VN was not due to enhanced cell adhesion or alterations in the cell cycle distribution; (d) both of the classic integrin receptors that bind VN (alpha(v)beta3, alpha(v)beta5) were capable of mediating this protective effect, because ligation of either of the two classic integrins conferred chemoresistance to Topo; and (e) chemoresistance observed with VN was associated with an increase in expression of two antiapoptotic proteins, Bcl-2 and Bcl-X(L), with a consequent increase in the ratios for Bcl-2:Bax and Bcl-X(L):Bax. VN, an ECM protein preferentially expressed at the tumor-brain interface in vivo, may confer a survival advantage to glioma cells at the advancing tumor margin and may thus, in part, underlie the high level of tumor recurrence at this interface.

Glioma invasion in vitro: regulation by matrix metalloprotease-2 and protein kinase C.

A hallmark of invasive tumors is their ability to effect degradation of the surrounding extracellular matrix (ECM) by the local production of proteolytic enzymes, such as the matrix metalloproteases (MMPs). In this paper, we demonstrate that the invasion of human gliomas is mediated by a 72 kDa MMP, referred to as MMP-2, and provide further evidence that the activity of MMP-2 is regulated by protein kinase C (PKC). The invasiveness of five human glioma cell lines (A172, U87, U118, U251, U563) was assessed in an in vitro invasion assay and was found to correlate with the level of MMP-2 activity (r2 = 0.95); in contrast, the activity of this 72 kDa metalloprotease was barely detectable in non-invasive control glial cells (non-transformed human astrocytes and oligodendrocytes). Treatment with 1,10-phenanthroline, a metalloprotease inhibitor, or with a synthetic dipeptide, containing a blocking sequence (ala-phe) specific for MMPs, resulted in a > 90% reduction in glioma invasion. Furthermore, this MMP-2 activity could be inhibited by the treatment of tumor cells with calphostin C, a specific inhibitor of PKC. Glioma cell lines treated with calphostin C demonstrated a dose-dependent decrease (IC50 = 30 nM) in tumor invasiveness with a concomitant reduction in the activity of the MMP-2. Conversely, treatment of non-invasive control astrocytes with a PKC activator (phorbol ester) led to a corresponding increase in their invasiveness and metalloprotease activity. These findings support the postulate that MMP-2 activity constitutes an important effector of human glioma invasion and that the regulation of this proteolytic activity can be modulated by PKC.

Apoptosis is induced in glioma cells by antisense oligonucleotides to protein kinase C alpha and is enhanced by cycloheximide.

The protein kinase C (PKC) activity of human glioma cells correlates with their rate of proliferation. We report here that the down-regulation of the predominant PKC isoform of glioma cells, alpha, by antisense phosphorothioate oligonucleotides (AS PTO) significantly reduced the rate of proliferation of three human glioma cell lines. This reduction in growth rate was attributed to apoptosis, as assessed by terminal deoxynucleotidyl transferase (TdT) assay. Unexpectedly, when low concentrations of the protein synthesis inhibitor cycloheximide (CHX) were administered to A172 cells immediately prior to AS PTO treatment, a marked enhancement in the number of apoptotic cells was observed. These findings suggest that PKC alpha plays a pivotal role in the ability of gliomas to avoid apoptotic cell death.

Mechanisms of glioma invasion: role of matrix-metalloproteinases.

One of the most lethal properties of high grade gliomas is their ability to invade the surrounding normal brain tissue, as infiltrated cells often escape surgical resection and inevitably lead to tumour recurrence. The consequent poor prognosis and survival rate underscore the need to further understand and target the cellular mechanisms that underly tumour invasiveness. Proteases which degrade the surrounding stromal cells and extracellular matrix proteins have been demonstrated to be critical effectors of invasion for tumours of both central and peripheral origin. Within the nervous system, the role of metalloproteinases as well as other classes of proteases in mediating the invasive phenotype of high grade gliomas has been an intense area of research. We present in this article a review of this literature and address the possibility that these proteases and the biochemical pathways that regulate their expression, such as protein kinase C, may represent potential targets in the therapy of high grade gliomas.

Protein kinase C and growth regulation of malignant gliomas.

This article reviews the role of the signal transduction enzyme protein kinase C in the regulation of growth of malignant gliomas, and describes how targetting this enzyme clinically can provide a novel approach to glioma therapy.

Protein kinase C in cultured adult human oligodendrocytes: a potential role for isoform alpha as a mediator of process outgrowth.

The extension of cellular processes from the oligodendrocyte soma is an early and critical event in myelin formation. Previous reports from this laboratory have implicated a role for protein kinase C (PKC) as an important intracellular mediator of this critical step in myelinogenesis. In the current study, the regrowth of fibers by adult human oligodendrocytes was examined and was found to be significantly enhanced by the PKC stimulator, 4 beta-phorbol-12,13-didecanoate (PDB); this was accompanied by a 400-500% increase in oligodendroglial PKC activity. In contrast to other cell types, the increased PKC activity in oligodendrocytes was not followed by subsequent down-regulation of the enzyme. The role of PKC in oligodendroglial process formation was further demonstrated by the ability of inhibitors of PKC to block the basal- or PDB-enhanced fiber outgrowth. As well, studies employing isoform-specific agonists implicated PKC alpha as the major determinant of fiber outgrowth by oligodendrocytes. The potential significance of PKC in myelin formation was further underscored by the observation that the synthesis of myelin basic protein, a prerequisite component for myelinogenesis, was increased by 2-fold in PDB-treated oligodendrocytes. Collectively, these observations suggest that PKC, in particular the alpha isoform, constitutes an important mediator in the initiation of myelin formation.

Antibody to beta-amyloid precursor protein recognizes an intermediate filament-associated protein in Alzheimer's and control fibroblasts.

The expression of beta-amyloid precursor protein (BAPP) and its mRNAs was studied in fibroblasts obtained from patients afflicted with Alzheimer's disease (AD) and age-matched controls. Using reverse transcriptase-polymerase chain reaction (RT-PCR), transcripts corresponding to 770, 751, 714, and 695 amino acids were detected in both AD and control fibroblasts. Antibody 22C11 against BAPP (Boehringer Mannheim) labeled an intracellular protein, specifically localized to the intermediate filament network. In addition to bands of the predicted molecular weights for BAPP (120-135 kDa), Western blotting revealed a 57 kDa band which was not evident in samples of human brain. As cytoskeletal elements are vital in maintaining cellular architecture and various cell interactions, localization of BAPP or a related molecule to the cytoskeleton suggests a possible structural role for this protein within the cell.

Staurosporine differentially inhibits glioma versus non-glioma cell lines.

We have previously demonstrated that the protein kinase C (PKC) activity of human glioma cell lines was significantly elevated (by 3 orders of magnitude) when compared to non-malignant adult human glia, and that the proliferation rate of several established human glioma cell lines correlated with the measured protein kinase C activity. The purpose of this study was to determine whether 1) elevated PKC activity was also a characteristic of fast growing cell lines of non-glial origin, 2) the proliferation rate of non-glioma cell lines correlated with their PKC activity and 3) the proliferation of non-glioma cell lines could be inhibited by staurosporine, a relatively selective PKC inhibitor, which significantly attenuates the growth of glioma cells. Eight established human non-glioma cell lines (bladder, colorectal, rhabdomyosarcoma-oligodendrocyte hybrid, melanoma, cervix, and fibroblast) were compared to the highly proliferative A172 glioma cell line. PKC activity was significantly higher in the glioma cell lines even though 3 of 8 of the non-glioma lines had higher proliferation rates than A172. In non-glioma cell lines, no correlation was found between the PKC activity and proliferation rates. Staurosporine was more effective in decreasing the proliferation of three glioma cell lines compared to the non-glioma cell lines. We conclude that PKC activity is differentially increased in glioma cell lines and that their proliferation rate is more sensitive to inhibition by staurosporine. Targetting the PKC system may prove to be of therapeutic benefit to patients with malignant gliomas.

Expression of the intermediate filament-associated protein related to beta-amyloid precursor protein is developmentally regulated in cultured cells.

It was previously reported that a monoclonal antibody to beta-amyloid precursor protein (mab22C11; Boehringer Mannheim, Indianapolis, IN) labels an intermediate filament-associated protein (beta APP-IFAP) in cultured human skin fibroblasts (Dooley et al.: J Neurosci Res 33:60-67, 1992). The time course of its expression and association with different classes of intermediate filaments has been assessed in neurons, Schwann cells, and astrocytes in dissociated cultures of murine brain and spinal cord-dorsal root ganglia; in primary cultures of human muscle; and in the epithelial cell line PtK1. beta APP-IFAP was expressed in all non-neuronal cell types examined. Mab22C11 immunoreactivity was minimal or absent following dissociation or subculture, but gradually increased with time. In fibroblasts, myoblasts, and epithelial cells, the distribution eventually resembled that of vimentin. With the exception of glial fibrillary acidic protein (GFAP), beta APP-IFAP was not associated with the intermediate filament proteins characteristically found in differentiated cells, i.e., desmin, the cytokeratins, and neurofilament proteins. No labeling of neurons by mab22C11 was observed at any stage of in vitro maturation. In sections of Alzheimer's brain, the antibody labeled a subpopulation of reactive astrocytes. It is suggested that beta APP-IFAP may be the product of a member of the beta APP multigene family expressed developmentally in non-neuronal cells.

Oligodendrocytes utilize a matrix metalloproteinase, MMP-9, to extend processes along an astrocyte extracellular matrix.

Matrix metalloproteinases (MMPs), the key effectors of extracellular matrix remodeling, have been demonstrated to regulate the extension of neurites from neuronal cell bodies. In this report we have addressed the hypothesis that oligodendrocytes (OLs) may utilize a similar mechanism in extending their processes during the initial phase of myelination. Furthermore, given our previous findings linking protein kinase C (PKC) to the OL process outgrowth, we tested the postulate that this signal transduction pathway may regulate MMPs and thus the process outgrowth phenotype. We demonstrate that in response to pharmacologic activators of PKC, cultured human OLs augment their process extension with a concomitant increase in the activity of an MMP, MMP-9, as measured by gelatin zymography. Similarly, the phorbol ester-enhanced process extension and increased MMP-9 activity were both inhibited by calphostin C, a selective PKC inhibitor. Also, MMP inhibitors such as 1,10-phenanthroline and synthetic dipeptides that inactivate the MMP catalytic site negated the 4beta-phorbol-12,13-dibutyrate (PDB)-mediated process extension, further supporting the key role of MMPs in process extension in vitro. Finally, the elevation of MMP-9 protein expression in the mouse corpus callosum, a tissue rich in OL and myelin, coincided with the previously documented temporal increase in myelination that occurs postnatally. Taken together, these data suggest that MMP-9 constitutes an important mediator of OL process outgrowth, and that this protease in turn can be regulated by PKC. The results are relevant not only to the initial steps of myelination during development, but also to the attempted remyelination that has been shown to occur in pathologic conditions such as MS.

Protein kinase C isoform alpha overexpression in C6 glioma cells and its role in cell proliferation.

Previous studies from this laboratory have demonstrated that protein kinase C (PKC) enzyme activity is highly correlated with the proliferation rate of glioma cells, and that glioma cells of both human and rat origin have very high PKC enzyme activity when compared to non-malignant glia including astrocytes, the antecedents of most gliomas. In the present study, by contrasting the rat C6 glioma cells with non-malignant rat astrocytes, we have sought to determine whether the high PKC enzyme activity of glioma cells was due to the overexpression of a specific isoform of PKC. By Western blot analyses, both C6 glioma cells and astrocytes were found to express PKC alpha, beta, delta, epsilon and zeta, but not gamma. Enzyme activity measurements revealed that the elevated PKC activity of glioma cells compared to glia was calcium-dependent, thereby implicating abnormal activity of the alpha or beta isoforms. On Western blots, when compared to astrocytes, glioma cells were determined to overexpress PKC alpha but not beta. An antisense oligonucleotide to PKC alpha, directed at the site of initiation of translation, inhibited the proliferation rate of glioma cells when compared to cells treated with control oligonucleotides; PKC enzyme activity and PKC alpha protein expression were significantly reduced by the antisense treatment. These results suggest that the high PKC enzyme activity of glioma cells, and its correspondence with proliferation rate, is the result of overexpression of isozyme alpha. Targetting PKC alpha in glioma cells may provide a refinement of therapy of glioma patients, some of which are already showing clinical stabilization when treated with drugs with PKC-inhibitory effects.

Antigen presentation by human fetal astrocytes with the cooperative effect of microglia or the microglial-derived cytokine IL-1.

Antigen presentation by endogenous glial cells is postulated to regulate reactivity of immune cells that gain entry into the CNS. We have previously observed, using a mixed lymphocyte reaction (MLR) system, that adult human-derived microglia can function as antigen-presenting cells (APC) for immediately ex vivo CD4+ T cells in a primary MLR (1 degree MLR) whereas astrocytes could not. We have now found that fetal human astrocytes can support CD4+ T cell proliferation in the presence of exogenous human recombinant (r) IL-2, and that astrocytes can support the continued proliferation of CD4+ T cells previously sensitized to sister astrocyte cultures in a secondary MLR. Additionally, adult human microglia, seeded into the nonpriming astrocyte: CD4+ T cell cocultures at non-T cell-stimulatory concentrations of 1000-5000 microglial cells per well, could reverse the inability of astrocytes to present antigen in the primary MLR. To examine the cellular basis for the inability of human astrocytes to function as APCs in the primary MLR, astrocyte- and microglial-enriched populations were established from human embryonic and adult brain, respectively, and analyzed for their ability to synthesize cytokines potentially relevant as accessory signals in the MLR. Microglia had transcript as determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) and protein as determined by bioassay for IL-1 alpha, IL-6, and TNF alpha. Human fetal astrocytes had transcript for IL-6 but not for IL-1 alpha or TNF alpha under basal culture conditions and following IFN gamma stimulation. The addition of human rIL-1 from 1-50 U/ml could reverse the inability of astrocytes to present antigen in the primary MLR. These studies demonstrate that although in vitro highly enriched cultures of astrocytes absent of microglia cannot present antigen to immediately ex vivo blood-derived CD4+ T cells in the MLR, in situ, with the cooperative help of microglia-derived cytokines or accessory surface molecules, astrocytes may function as central nervous system APCs.

Interferon beta-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9.

In multiple sclerosis (MS), the influx of activated T lymphocytes into the brain parenchyma leads to the subsequent damage of oligodendrocytes, the cells that produce central nervous system (CNS) myelin. We report here that interferon beta-1b (IFNbeta-1b), a drug shown to be efficacious in the treatment of patients with MS, decreases the in vitro migration of activated T lymphocytes through fibronectin (FN), a major component of the basement membrane that surrounds cerebral endothelium. At 1,000 IU/ml, IFNbeta-1b reduced the migratory rate to that of unactivated T cells. In contrast, IFNgamma at 1,000 IU/ml, which caused a similar decrease (25%) in the proliferation rate of T lymphocytes as IFNbeta-1b, did not affect migration. All T-lymphocyte subsets and natural killer (NK) cells were demonstrated by flow cytometry to be equally affected by IFNbeta-1b treatment. 125I-Western blot analyses revealed that IFNbeta-1b treatment resulted in a marked reduction of the ability of T cells to cleave FN. The substrate-degrading capability of T lymphocytes was shown to be due predominantly to the activity of a 92-kd matrix metalloproteinase, MMP-9, whose levels were decreased by IFNbeta-1b. We suggest that the clinical benefits of IFNbeta-1b treatment in MS patients may be in part a result of the ability of this drug to significantly decrease MMP-9 activity, leading to a reduction of T-lymphocyte infiltration into the CNS.

Migratory behavior of lymphocytes isolated from multiple sclerosis patients: effects of interferon beta-1b therapy.

Previous reports by our group and by others have shown that in vitro treatment of T cells derived from healthy, normal subjects with interferon beta-1b (IFN-beta1b) reduces metalloproteinase (metalloproteinase type 9 [MMP-9]) activity with a consequent reduction in lymphocyte migration. In this study, we used a Boyden chamber assay to assess the migratory capacity of T cells derived from multiple sclerosis patients who either did or did not receive IFN-beta1b. Lymphocytes derived from patients treated for less than 2 years with IFN-beta migrated at a low rate that was indistinguishable from that of cells isolated from healthy donors. However, longer term treatment with IFN (>3.5 years) was associated with a reversion of the migration to a high level that did not differ statistically from that of cells isolated from untreated multiple sclerosis patients. For both high-migratory groups, migration could be reduced to control levels after the exogenous addition of TIMP-1, a relatively specific inhibitor of the MMP-9, implicating this protease in the process of T-cell migration. Our findings suggest that one of the mechanisms by which IFN-beta exerts its action is by reducing MMP-9 activity and thus the entry of inflammatory cells into the nervous system and, as such, MMPs may constitute potential therapeutic targets in inflammatory diseases such as multiple sclerosis.