The expression of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (E-NPP1) is correlated with astrocytic tumor grade

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Abstract

Objective

Astrocytic brain tumors are subdivided in four grades. The most aggressive and invasive one is grade IV or glioblastoma multiforme (GBM). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (E-NPP1), a membrane-bound enzyme, is involved in many cellular processes such as modulation of purinergic signalling, nucleotide recycling, regulation of extracellular pyrophosphate levels and stimulation of cell motility. In this study, the use of anti-NPP1 antibody in the determination of astrocytic tumor grade is evaluated.

Materials and methods

Paraffin-embedded surgical specimens from 41 primary human astrocytic brain tumors (grade I = 2; grade II = 10; grade III = 9; grade IV = 20) and 5 control samples are immunostained against NPP1 and glial fibrillary acid protein an astrocytic marker.

Results

In this communication, we report the expression of NPP1 in human astrocytic brain tumors. No expression could be detected in control tissue. We observed a remarkable up regulated expression of NPP1 in GBM. Taking the latter as 100%, grade I has a relative NPP1 staining of 7%, whereas grade II and III have a similar NPP1 expression level of 53% and 47% respectively.

Conclusion

A correlation is found between the up-regulated expression of NPP1 and the grade of the astrocytic tumor. Further investigation of NPP1 expression, especially in GBM, is necessary to determine the role of NPP1 in astrocytic brain tumor progression.

Introduction

Astrocytic brain tumors or astrocytomas are the most common glioma, accounting for about half of all primary brain tumors. Astrocytomas are glial cell tumors, derived from star-shaped glial cells that can occur in most parts of the brain. Astrocytomas are of two main types: (i) high-grade and (ii) low-grade tumors [1]. The first group grows rapidly and can easily spread through the brain. They are aggressive and require very intensive therapy. The second group is usually localized and grow slowly over a long period of time. The World Health Organization (WHO) classification for astrocytomas is based on cellular origin and histological appearance. Astrocytomas are divided into four grades: (i) the low-grade tumors consist of WHO grade I (pilocytic astrocytoma) and WHO grade II (low-grade astrocytoma) and (ii) the high-grade tumors consist of WHO grade III (anaplastic astrocytoma) and WHO grade IV (glioblastoma multiforme (GBM)) [2], [3], [4]. Low-grade astrocytomas are one of the least common brain tumors, while high-grade astrocytomas are the most common malignant brain tumors in adults [2]. GBM is a highly aggressive tumor that originates from the astrocytic cells of the brain [5]. Primary brain tumors occur in all age groups, but the incidence increases in elderly patients [2]. The average survival time of patients with GBM is less than one year after diagnosis due to resistance of the tumor to therapeutic interventions [5]. However, GBM patients below the age of 50 years have a better prognosis than older individuals [6].

In vitro, rat C6 glioma cells are often used as an experimental model system for the study of biochemical properties of astrocytes and glioblastoma growth and invasion. These cells originated from brain tumors induced in Wistar–Furth rats by exposure to N,N′-nitroso-methylurea [7]. The cell line is characterized by proliferative and invasive properties due to point mutations in PTEN resulting in a constitutively active PI 3K/PKB-signalling pathway [7], [8]. We previously observed the expression of an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), identified as NPP1, on the membrane of C6 cells. No other members of the E-NPP family were demonstrated in these cells [9]. Originally, Takahashi et al. [10] discovered NPP1 as the plasma cell differentiation antigen PC-1 on the surface of mouse B-lymphocytes. They also found that this glycoprotein was present in homogenates of liver, brain and kidney [11].

E-NPPs are membrane-bound ecto-enzymes that are involved in multiple physiological mechanisms such as nucleotide recycling, modulation of purinergic signalling, regulation of extracellular pyrophosphate levels and stimulation of cell motility [11]. These E-NPPs belong to a multigene family with seven members. NPP1 and NPP3 are type II transmembrane glycoproteins with a short intracellular amino-terminal domain, a single transmembrane domain and a large extracellular carboxy-terminal part. NPP2 is not a transmembrane protein but is synthesized as a pre-pro-enzyme, which is secreted after removal of the N-terminal signal peptide [12]. NPP1 and NPP3 have a broad substrate specificity and release nucleoside 5′-monophosphate from a variety of nucleotides and their derivates e.g. NPP1 converts ATP into AMP and PPi and can therefore affect purinergic signalling [11], [13], [14], [15]. NPP2 is an extracellular lysophospholipase D [16]. The other E-NPP members, NPP4-7, are type I transmembrane glycoproteins with a short intracellular carboxy-terminus and a significant smaller extracellular domain as compared to NPP1-3 [11], [13]. The functions of NPP4-5 are unknown while NPP6 and NPP7 hydrolyse choline phosphate esters [17], [18]. Although molecular and structural characteristics of E-NPPs are intensively investigated, their functional role and distribution in the brain and other organs is still controversial.

In this study, we will exclusively investigate astrocytomas and GBM. We measured the expression of NPP1 in human astrocytic brain tumors and correlated its increased expression with the histological grade of astrocytomas.

Section snippets

Tumor samples

The protocols and procedures were approved by the ethical committee of the University Hospital of Antwerp (A02-003). Surgical specimens of astrocytomas were obtained from patients treated between 1992 and 2003 in the department of Neurosurgery of Middelheim General Hospital (ZNA) of Antwerp, Belgium. Astrocytomas were classified according to the WHO classification system into low-grade astrocytoma (grades I–II), anaplastic astrocytoma (grade III), or glioblastoma multiforme (grade IV).

Results

Previous findings in our laboratory demonstrated NPP1 expression in rat C6 glioma cells [14], a model system for GBM and astrocytes. Hence, we expected expression of NPP1 in human astrocytic brain tumors. The latter supposition is demonstrated by immunohistochemistry on tissue sections of astrocytomas.

In this study, we only investigated astrocytomas and GBMs. Brain samples of 46 patients, 11 women and 35 men, were tested by immunohistochemistry for the presence of GFAP and NPP1. Of these

Discussion

Nucleotides are important signalling molecules regulating short-term and long-term cellular functions in the central nervous system [20], [21]. Nucleotide-dependent signalling is modulated by hydrolysis of nucleotides by cell surface-bound enzymes including E-NPP [11], [15], [22]. Many tissues contain several NPP subtypes that are functional in physiological and biochemical processes such as bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor

Acknowledgments

We thank Prof. Dr. J. Goding of the Department of Pathology and Immunology, Monash Medical School, Monash University (Australia) for the gift of anti-NPP1 antibodies and antigens. We also acknowledge the technical assistance of U. Lübke, a member of the Biobank facility of the Institute Born-Bunge (University of Antwerp, Belgium) for her immunohistochemical skills and experience. Also we thank the central Biobank facility of the Institute Born-Bunge for all brain tissue material. Prof. Dr. A.

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