Infiltration of the central nervous system by neoplastic cells in patients with glioblastoma multiforme (GBM) leads to neurological dysfunction and eventually to death. The elucidation of the mechanisms underlying the aggressive nature of GBM aims at improving radio-, chemo- and gene therapy. This review is focused on the use of rat C6 glioma as an experimental model system for GBM and provides an overview of the experimental data published in the literature using this cell line in elucidating the mechanism of tumor growth, angiogenesis and invasion, and in the design and evaluation of anticancer therapies. Understanding the stages of malignant brain tumor progression requires a series of experimental approaches with a varying degree of complexity. Implantation of malignant cells into animal brain tissue closely resembles in vivo tumor growth and has the advantage over simplified models that inflammatory and vascular mechanisms are activated. However, the complexity of these models makes it difficult to identify the individual processes involved in sustained tumor growth, angiogenesis and invasion. In cell culture models, the effect of growth factors, extracellular matrix components, proteases and adhesion molecules can be investigated. The secretion of tumor-derived factors into the medium can also be analyzed when simplified models are used. This review is a compilation of experimental data focused on the characterization of tumor-related processes and on the evaluation of new therapies for the treatment of malignant glial neoplasms using rat C6 glioma as a model system.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.

Many cellular processes have been identified in which phosphatidylinositol 3-kinase has a key regulatory function. As an oncogene, it is also involved in the development of cancer. The transformation and progression of normal cells towards an advanced stage tumor and/or towards metastatic lesions involves a complex series of events, including genetic alterations, leading to aberrant cell cycle progression, altered adhesion and motility characteristics, inhibition of apoptosis and induction of angiogenesis.This review highlights the processes involved in the pathogenesis of cancer in which phosphatidylinositol 3-kinase is involved and provides an overview of the possible mechanisms by which the enzyme exerts its oncogenic action.Keywords: angiogenesis; apoptosis; cancer; cell cycle; invasion; metastasis; motility; oncogenic transformation; phosphatidylinositol 3-kinase; proliferation.Over the past 10 years, research on phosphatidylinositol 3-kinases has demonstrated that this family of enzymes contains important regulators of cellular signaling. They are activated by G-protein-coupled receptors or receptors with an intrinsic or associated protein tyrosine kinase activity and/or proteins that are tyrosine phosphorylated in response to extracellular stimuli [1]. Another way in which phosphatidylinositol 3-kinases are activated is by a direct interaction with the small GTPase Ras [2,3]. Upon activation, these enzymes phosphorylate inositol lipids at the D-3 position of the inositol ring to generate the 3-phosphoinositides, phosphatidylinositol 3-phosphate [PtdIns(3)P], phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. On the basis of their primary structure, regulation and in vitro lipid substrate specificity, three classes of phosphatidylinositol 3-kinases have been defined. Class I phosphatidylinositol 3-kinases generate PtdIns(3)P, PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 and consist of four mammalian p110 catalytic isoforms (p110a, b, g and d) which associate with the p85-family of regulatory subunits, except for p110g which binds to a p101 adaptor [4±6]. The phosphatidylinositol 3-kinases of class II generate PtdIns(3)P and PtdIns(3,4)P 2 and are large (170±210 kDa) proteins which all contain a characteristic C-terminal region with homology to C2 domains [7]. The class III enzymes only produce PtdIns(3)P and only contain a catalytic and a phosphoinositide kinase domain [7,8]. Phosphoinositides are functional as second messengers and serve as binding targets for proteins containing pleckstrin-homology domains or FYVE-fingers [9,10]. Examples of pleckstrin-homologydomain-containing proteins, which preferentially bind to PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 ] are the serine/threonine kinase Akt (also known as protein kinase B), 3-phosphoinositide-dependent kinase-1, guanine nucleotide exchange factors, certain Bruton's tyrosine kinase family members and phospholipase Cg (PLCg). In contrast, FYVE-fingercontaining proteins like early endosome antigen ...

Abstract:The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the ␣-and ␥-phosphates. The enzyme degraded ATP into AMP and PP i and, depending on the ATP concentration, accounted for ϳ50 -75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 M abolished the degradation of ATP into AMP and PP i . The nucleotide pyrophosphatase has an alkaline pH optimum and a K m for ATP of 17 Ϯ 5 M. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3Ј,5Ј-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.