Elsevier

Brain Research

Volume 1352, 17 September 2010, Pages 200-207
Brain Research

Research Report
Expression of targeting protein for Xenopus kinesin-like protein 2 is associated with progression of human malignant astrocytoma

https://doi.org/10.1016/j.brainres.2010.06.060Get rights and content

Abstract

In humans, the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cell cycle-associated protein, and altered TPX2 expression has been found in various malignancies. However, the contribution of TPX2 expression to astrocytoma progression is unclear. The aim of this study was to investigate TPX2 expression in human astrocytoma samples and cell lines. TPX2 protein expression was detected in the nucleus of astrocytoma tissues by immunohistochemistry and immunofluorescence staining. Real-time PCR and Western blot analysis showed that the expression levels of TPX2 were higher in high-grade astrocytoma tissues and cell lines than that in low-grade astrocytoma tissues and normal cell lines. Immunohistochemical analysis of tumor tissues from 52 patients with astrocytoma showed that TPX2 over-expression was significantly associated with decreased patient survival. In addition, down-regulation of the TPX2 gene by RNA interference inhibited proliferation of U87 cells. TPX2 gene silencing also increased early-stage apoptosis in U87 cells. Western blotting and real-time PCR showed changes in the protein and mRNA expression of Aurora A, Ran, p53, c-Myc and cyclin B1 in U87 cells that had been transfected with pSUPER/TPX2/siRNA. These data suggest that TPX2 expression is associated with the progression of malignant astrocytoma.

Research Highlights

►Demonstrated that a gene known as targeting protein for Xenopus kinesin-like protein 2 (TPX2) may serve as an attractive anticancer target for malignant astrocytoma. The high expression of TPX2 expression in malignant astrocytoma tissues and cells may be associated with the progression of malignant astrocytoma. This result may offer a novel approach to controlling malignant astrocytoma.

Introduction

Malignant astrocytomas are the most common form of primary brain tumor, of which glioblastoma multiforme (GBM) is the most frequent malignant type (Levin, 1999). Despite improvement in treatment strategies, the prognosis for patients with malignant astrocytoma remains poor (Wechsler-Reya and Scott, 2001). Multiple genetic aberrations have been described in malignant astrocytoma, including amplification, homozygous deletion and the mutation of multiple genes (Ichimura et al., 2000, Riemenschneider et al., 2003, Ohgaki and Kleihues, 2007, Balss et al., 2008, Parsons et al., 2008). Several important pathways involved in the regulation of cell division are disrupted in glioblastoma (Cancer Genome Atlas Research Network, 2008). With an improved understanding of the molecular pathogenesis of malignant astrocytoma, targeted molecular therapies have recently evolved (Chi and Wen, 2007, Ohgaki and Kleihues, 2007, Sathornsumetee et al., 2007). The identification of additional astrocytoma-associated tumor markers is thus important for further research on astrocytoma pathogenesis. The targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cell cycle-associated human protein (Heidebrecht et al., 1997, Zhang et al., 1999) and its expression is tightly regulated by the cell cycle. The aberrant expression of TPX2 has been found in various malignant tumors (Bonatz et al., 1999, Hufton et al., 1999, Rudolph et al., 1999, Gruss et al., 2002, Krams et al., 2003, Tonon et al., 2005, Shigeishi et al., 2009, Kadara et al., 2009, Warner et al., 2009, Satow et al., 2010). These results suggest that TPX2 plays a role in the oncogenesis of some malignancies. However, the expression patterns of TPX2 in astrocytoma are unclear. In the present study, we investigated the expression of TPX2 in human astrocytoma and related cell lines in order to test whether a correlation existed between TPX2 protein levels and the grade of cell differentiation. In addition, we evaluated the proliferation and apoptosis of cells when the TPX2 gene was inhibited by RNA interference in order to examine the relationships between TPX2 and astrocytoma.

Section snippets

Expression of TPX2 in astrocytoma tissue

Positive TPX2 immunoreactivity was detected in the nuclear compartments of 35 out of 52 astrocytoma tissue samples tested, while no positive TPX2 staining was detected in the normal brain tissue (Table 1, Fig. 1A). Analysis of the positive-staining data showed that the TPX2 expression level in high-grade astrocytomas was significantly higher than that in low-grade astrocytomas (P = 0.045). Similar results were also obtained by real-time PCR (P < 0.001) and Western blot analysis (P = 0.003) (Fig. 1B

Discussion

In the present study, we present both clinical and experimental evidence that TPX2 expression affects the progression of human malignant astrocytoma. The mitotic spindle apparatus segregates and redistributes chromosomes during mitosis (Sharp et al., 2000) and is composed of a highly dynamic microtubule skeleton that is associated with a variety of proteins, including kinesin-like and microtubule-associated proteins (Desai and Mitchison, 1997). As a cell microtubule and cycle-associated human

Patients and tissue samples

Samples were obtained from 52 patients with newly diagnosed primary astrocytoma (5 PA, WHO grade I; 16 DA, WHO grade II; 13 AA, WHO grade III; 18 GBM, WHO IV), who had not received therapy before sample collection. Samples were collected between 2004 and 2006 in Changzheng Hospital, the Second Military Medical University (Shanghai, China). The research protocol was approved by the Ethics Committee of Second Military Medical University. All samples were confirmed by pathological examination

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    1

    Bin Li and Xiang-Qian Qi contributed equally to this work.

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