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Identification of differentially expressed proteins in human glioblastoma cell lines and tumors
Rulin Zhang1, Tammy-Lynn Tremblay, Angela McDermid
1Institute for Biological Sciences, National Research Council, Ottawa, Canada.
Abstract:
An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGdeltaEGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGdeltaEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors.
Insights
A common epidermal growth factor receptor (EGFR) mutation in glioblastoma drives malignancy. Researchers identified specific proteins upregulated in glioblastoma, suggesting potential diagnostic markers for this aggressive brain tumor.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Glioblastoma multiforme (GBM) is an aggressive brain tumor.
- A specific type III mutation in the epidermal growth factor receptor (EGFR) is frequently observed in primary GBM.
- This EGFR mutation is associated with increased tumor cell malignancy.
Purpose of the Study:
- To investigate differential protein expression in glioblastoma cells with and without the type III EGFR mutation.
- To identify potential protein biomarkers for glioblastoma diagnosis and understanding tumor behavior.
Main Methods:
- Proteomic analysis using two-dimensional gel electrophoresis, LC-MS-MS, and MALDI-TOF MS.
- Differential protein expression profiling of fetal human astrocytes (FHA), U87MG cells, and U87MG cells with EGFR deletion (U87MGdeltaEGFR).
- Validation of candidate protein markers (Hsp27, major vault protein, tissue transglutaminase, cystatin B) using Western blot, ELISA, and RT-PCR in various brain tissue samples.
Main Results:
- 23 proteins were exclusively or highly expressed in FHA, and 29 in U87MG cells.
- Ubiquitin, cystatin B, and tissue transglutaminase were upregulated in U87MGdeltaEGFR cells.
- Hsp27, major vault protein, tissue transglutaminase, and cystatin B were highly expressed in GBM tissues compared to nonmalignant brain tissues.
Conclusions:
- The identified proteins (Hsp27, major vault protein, tissue transglutaminase, cystatin B) show promise as diagnostic markers for glioblastoma.
- These proteins may also serve as functional markers related to glioblastoma invasiveness and drug resistance.
