Proteomic identification of glutamine synthetase as a differential marker for oligodendrogliomas and astrocytomas

Laboratory investigation

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Object

Astrocytomas and oligodendrogliomas are primary CNS tumors that remain a challenge to differentiate histologically because of their morphological variability and because there is a lack of reliable differential diagnostic markers. To identify proteins that are differentially expressed between astrocytomas and oligodendrogliomas, the authors analyzed the proteomic expression patterns and identified uniquely expressed proteins in these neoplasms.

Methods

Proteomes of astrocytomas and oligodendrogliomas were analyzed using 2D gel electrophoresis and subsequent computerized gel analysis to detect differentially expressed proteins. The proteins were identified using high-performance liquid chromatography accompanied by tandem mass spectrometry. To determine the role of the differentially expressed proteins in astrocytes, undifferentiated glial cell cultures were treated with dibutyryl–cyclic adenosine monophosphate (cAMP).

Results

Two-dimensional gel electrophoresis revealed that glutamine synthetase was differentially expressed in astrocytomas and oligodendrogliomas. Western blot and immunohistochemical analyses confirmed the increased expression of glutamine synthetase in astrocytomas compared with oligodendrogliomas. Whereas glutamine synthetase expression was demonstrated across all grades of astrocytomas (Grade II–IV [15 tumors]) and oligoastrocytomas (4 tumors), it was expressed in only 1 oligodendroglioma (6% [16 tumors]). Treatment of undifferentiated glial cell cultures with dibutyryl-cAMP resulted in astrocyte differentiation that was associated with increased levels of glial fibrillary acidic protein and glutamine synthetase.

Conclusions

These data indicate that glutamine synthetase expression can be used to distinguish astrocytic from oligodendroglial tumors and may play a role in the pathogenesis of astrocytomas.

Abbreviations used in this paper: cAMP = cyclic adenosine monophosphate; GFAP = glial fibrillary acidic protein; HPLC = high-performance liquid chromatography; PAGE = polyacrylamide gel electrophoresis; SDS = sodium dodecyl sulfate.

Article Information

* Drs. Zhuang and Qi contributed equally to this work.

Address correspondence to: Zhengping Zhuang, M.D., Ph.D, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 10 Center Drive, Building 10, Room 3D20, Bethesda, Maryland 20892-1414. email: zhuangp@ninds.nih.gov.

Please include this information when citing this paper: published online June 17, 2011; DOI: 10.3171/2011.5.JNS11451.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Proteomic comparison through 2D-PAGE. The proteomes of 3 WHO Grade II oligodendrogliomas and astrocytomas were resolved using 2D-PAGE and visualized through silver staining. Two representative gels are displayed. Arrowheads highlight differential proteins found across all samples analyzed.

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    Schematic of glutamine synthetase reaction in the CNS. When expressed in astrocytes, glutamine synthetase is responsible for converting extracellular glutamate into glutamine through adenosine triphosphate hydrolysis and amination. This reaction is critical for ammonia metabolism in the CNS and also plays an important role in the recycling of glutamate as a neurotransmitter or production of glutamine for other metabolic or signaling activities.

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    Western blot analysis comparing 3 WHO Grade II oligodendrogliomas and astrocytomas. A: Glutamine synthetase is expressed at higher levels in the astrocytomas. B: Glutamine synthetase expression is maintained across astrocytomas spanning WHO Grades II–IV.

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    Immunohistochemical results. A: When staining normal brain, glutamine synthetase has a diffuse staining pattern. However, within astrocytomas, glutamine synthetase staining reveals individual cell bodies and stains a majority of the tumors cells within the sample. Little to no staining was observed in the majority of oligodendrogliomas. B: Within serially cut sections of formalin-fixed paraffin-embedded mixed oligoastrocytomas, glutamine synthetase staining was able to resolve individual tumor cells better than GFAP and also demonstrated higher selectivity in excluding tumor cells that had morphological traits traditionally associated with neoplastic oligodendroglia (row 2, column 3). Original magnification × 10.

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    A: In murine primary astrocyte progenitor cells, glutamine synthetase (GS) is expressed in higher levels than GFAP and its level increases after cells are prompted to differentiate through treatment with dibutyryl-cAMP (dbcAMP). B: Immunofluorescent staining showing that glutamine synthetase and GFAP are highly expressed in terminal differentiated astrocytes. Bar = 10 mm. C: Immunofluorescent staining demonstrating glutamine synthetase widely expressed in glial cells in brain, whereas GFAP expression is only seen in a small population of glial cells. Bar = 50 μm.

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