Voon Wee Yong

Enhanced protein kinase C activity correlates with the growth rate of malignant gliomas in vitro.

Direct measurement of protein kinase C (PKC) activity in vitro revealed a significant increase in the activity of the enzyme in all human malignant glioma lines examined and the rat C6 tumor in comparison with control nonneoplastic astrocyte and mixed glial cultures. The total and particulate PKC activity in these cell types correlated strongly [r = 0.98 (P less than 0.001) and 0.94 (P = 0.002), respectively] with the maximal growth rates as measured by 3H-thymidine incorporation in each of the samples. An alteration in the growth rate of an individual glioma line (A172) by varying the serum concentration in the growth medium produced comparative changes in the measured PKC activity. The addition of the phorbol ester phorbol-12-myristate-13-acetate to this tumor line under high serum conditions produced down-regulation of the enzyme, which was accompanied by a corresponding reduction in thymidine incorporation. The administration of the PKC inhibitor staurosporine produced a dose-related decrease in the basal proliferation rate of glioma lines A172 and C6, as measured by 3H-thymidine uptake and confirmed by flow cytometry, indicating that the high intrinsic PKC activity is amenable to pharmacological manipulation. Cytofluorometric deoxyribonucleic acid cell cycle analysis of the tumors treated with PKC modulators demonstrated that reduced proliferation rates were caused by an inhibition of entrance into the deoxyribonucleic acid synthesis (S) phase (decrease in proliferative index), supporting the evidence that these modulators are not slowing the tumor growth in a nonspecific cytotoxic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

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 anin 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.

Protein Kinase C Activity Correlates with the Growth Rate of Malignant Gliomas: Part II. Effects of Glioma Mitogens and Modulators of Protein Kinase C

The proliferation rates of gliomas may be modulated by the protein kinase C (PKC) signal transduction system. The present study was undertaken to further examine the role of PKC system in growth regulation of gliomas in vitro by measurement of PKC activity over various phases of tumor growth and by assessing its potential role as a signal transduction system induced by serum mitogens and the known glioma mitogens epidermal growth factor and fibroblast growth factor. All human glioma lines examined, and the rat glioma C6, displayed high PKC activity relative to nonmalignant glial cells, which correlated with their proliferation rates over their respective growth phase. Frozen surgical human malignant glioma specimens also displayed high PKC activity. The relatively selective PKC inhibitor staurosporine (SP) reduced PKC activity and corresponding growth rates in a dose-related manner. Stimulation of PKC with phorbol esters under different concentrations of serum in the growth medium indicated that the high PKC activity, which correlated with their rapid growth rates, is highly susceptible to down-regulation by these agents. Epidermal growth factor and fibroblast growth factor increased both PKC activity and the growth rate of glioma line A172; addition of SP reduced the growth rate to levels observed in SP-treated control tumors, indicating that PKC may be a common signal transduction system induced by these mitogens. These results implicate PKC as an important signal transduction system regulating glioma growth, and offers a potential target for tumor inhibition.

Interferon-β is a potent promoter of nerve growth factor production by astrocytes

Abstract: Recent clinical evidence has suggested that interferon‐β is efficacious in the treatment of the demyelinating disease, multiple sclerosis. The mechanism of its efficacy remains unclear, and suggested modes of action have focused on immune modulation. Nonimmune effects of interferon‐β may also contribute to its efficacy. Given that astrocytes produce a range of neurotrophic factors, we examined the possibility that interferon‐β could increase the astrocytic production of nerve growth factor (NGF), which has been reported to cause oligodendrocytes to proliferate and to extend their processes; these phenotypes can impact favorably on remyelination. When the recombinant form of mouse interferon‐β was added to mouse astrocyte cultures, a dose‐dependent increase in NGF mRNA was obtained. The 40‐fold increase in NGF mRNA elicited by 1,000 U/ml interferon‐β was far more potent than that produced by other NGF‐elevating agents in this study. In concordance, the protein for NGF was elevated by interferon‐β. The production of NGF by interferon‐β may be relevant to its clinical efficacy in multiple sclerosis. Furthermore, we suggest the potential utility of interferon‐β in Alzheimers disease.

Astrocytes promote process outgrowth by adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix

Cell‐cell interactions regulate many important functions within the central nervous system. In this report, we demonstrate that process outgrowth by adult human oligodendrocytes (OLs) in vitro, an early event of myelinogenesis in vivo, is promoted by astrocytes. To elucidate the mechanisms by which astrocytes might exert this effect, we tested several growth factors known to be produced by astrocytes and found that only basic fibroblast growth factor (bFGF) could enhance process extension by the OL. In correspondence, the treatment of astrocytes with a neutralizing antibody to bFGF decreased their effects in promoting oligodendroglial process outgrowth. The potency of bFGF, however, was only one‐third that of astrocytes, and since bFGF did not synergize with other soluble growth factors, we investigated the potential facilitatory role of the extracellular matrix (ECM) deposited by astrocytes. The astrocyte ECM was found to be a promoter of oligodendroglial process extension, and significantly, bFGF synergized with astrocyte ECM to match the potency of live astrocytes. The astrocyte ECM was found in Western blot analyses to contain fibronectin, vitronectin, and laminin. These purified ECM components, as well as heparan sulfate proteoglycan, did not promote oligodendroglial process extension by themselves, although laminin and fibronectin potentiated the effects of bFGF. We conclude that process outgrowth by OLs is guided by astrocytes; the mechanism of the astrocyte effect appears to be due to the combination of bFGF and an unidentified ECM component.

Astrocyte reactivity in neonatal mice : Apparent dependence on the presence of reactive microglia/macrophages

In neonatal mice, an acute injury produced by a stab wound to the cortex results in minimal astrocyte reactivity, as has been observed by others. However, if the source of the stab wound, a piece of nitrocellulose (NC) membrane, were now implanted in the cortex for a period of time (chronic NC implant injury), then extensive astroglial reactivity in the neonatal brain ensues. The astrogliosis is manifested by increased mRNA, protein content, and immunoreactivity for GFAP, and by ultrastructural changes. Given the previous reports that inflammatory cytokines are possible mediators of astrocyte reactivity (e.g., Balasingam et al: J Neurosci 14:846, 1994), we examined the brain parenchyma of neonatal mice following an NC stab or implant injury, with minimal or extensive astrogliosis, respectively, for a possible differential representation of inflammatory cells. A significant correlation (r = 0.87, P < 0.05) was observed between the occurrence of astrogliosis and the presence of reactive microglia/macrophages; no other inflammatory cell type was detected in the brain parenchyma of neonatal mice following NC implant injury. We suggest that reactive microglia/macrophages are required for the evolution of cells into reactive astrocytes following insults to the neonatal brain.

Astrocytes and catalase prevent the toxicity of catecholamines to oligodendrocytes

Metabolism of catecholamines can generate reactive free radical species, including hydrogen peroxide (H2O2), that are potentially harmful to cells. In this study, norepinephrine (NE) and epinephrine (EPI) were found to be toxic to oligodendrocyte (OL) cultures derived from adult rat brain. The catecholamine toxicity, reproduced by equimolar concentrations of H2O2, could be completely prevented by simultaneous treatment of OLs with the H2O2-decomposing enzyme catalase. These results implicate H2O2 produced by metabolism of NE and EPI as the toxic intermediate. Since OLs in vivo are not normally susceptible to the toxicity of catecholamine neurotransmitter molecules, we sought to examine the involvement of another cell type closely apposed to OL, that is astrocytes, as a protectant against catecholamine toxicity. When adult rat OLs were seeded onto a monolayer of neonatal rat astrocytes, the toxicity of NE, EPI and H2O2 to OLs was completely prevented; medium conditioned by astrocytes did not prevent the manifestation of H2O2 toxicity on OLs. We conclude that the OL-myelin complex is vulnerable to free radical-mediated damage, especially when the protective functions of astrocytes are impaired.

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

SummaryPrevious 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 PKCα, β, δ, ɛ and ζ, but not γ. Enzyme activity measurements revealed that the elevated PKC activity of glioma cells compared to glia was calcium-dependent, thereby implicating abnormal activity of the α or β isoforms. On Western blots, when compared to astrocytes, glioma cells were determined to overexpress PKCa but not β. An antisense oligonucleotide to PKCα, 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 PKCa 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 a. Targetting PKCa 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.

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.

Signal transduction for proliferation of glioma cells in vitro occurs predominantly through a protein kinase C-mediated pathway.

Previous work has demonstrated that glioma cells have very high protein kinase C (PKC) enzyme activity when compared to non-malignant glia, and that their PKC activity correlates with their proliferation rate. The purpose of this study was to determine whether the elevated PKC activity in glioma is secondary to an autonomously active PKC isoform implying oncogenic transformation, or whether this activity is driven by upstream ligand-receptor tyrosine kinase interactions. We treated established human glioma cell lines A172, U563 or U251 with either the highly selective PKC inhibitor CGP 41 251, or with genistein, a tyrosine kinase inhibitor. The proliferation rate and PKC activity of all the glioma lines was reduced by CGP 41 251; the IC50 values for inhibiting cell proliferation corresponded to the IC50v values for inhibition of PKC activity. Genistein also inhibited cell proliferation, with IC50 proliferation values approximating those for inhibition of tyrosine kinase activity in cell free protein extracts. Importantly, in genistein-treated cells, downstream PKC enzyme activity was dose dependently reduced such that the correlation coefficient for effects of genistein on proliferation rate and PKC activity was 0.92. These findings suggest that upstream tyrosine kinase linked events, rather than an autonomously functioning PKC, result in the high PKC activity observed in glioma. Finally, fetal calf serum (FCS) evoked a strong mitogenic effect on glioma cell lines. This mitogenic activity was completely blocked by CGP 41 251, suggesting that although the many mitogens in FCS for glioma cells signal initially through genistein-inhibitable tyrosine kinases, they ultimately channel through a PKC-dependent pathway. We conclude that proliferative signal transduction in glioma cells occurs through a predominantly PKC-dependent pathway and that selectively targeting this enzyme provides an approach to glioma therapy.