[Analysis of the water-soluble protein fraction of glioma cells by two-dimensional electrophoresis].
The authors analyzed water-soluble proteins of culture human and rat glioma cells by two-dimensional polyacrylamide gel electrophoresis methods. Glioma cells were suspended in distilled water and then destroyed by freezing and thawing to obtain the water-soluble protein fraction. A modification of O'Farrell's non-equilibrium pH gradient (NEPHGE) method was used to analyze differences in protein mapping. Manabe's microscale two-dimensional electrophoresis without denaturing agents was used to detect proliferating cell nuclear antigen (PCNA/cyclin) by Western blotting. With O'Farrell's NEPHGE method and silver staining, at least 200 different polypeptides were clearly identified in each cell line. Cytoskeletal proteins, such as actin, were consistently separated in all cell lines. Marked differences in the protein map were observed between human and rat glioma cell lines, and even within the same species. Presumably, these differences are attributable to cell-biological difference in the glioma cells lines. Some proteins that were prominent in proliferating cells were scant in the protein maps of cells cultured for 24 hours in medium not containing calf serum, which suppresses cell growth. PCNA, an acidic nuclear protein that appears only in the late G1-S phase and is believed to be involved in cell proliferation, was detected by Western blotting and indirect immunostaining. Quantitative analysis of PCNA spots on the protein map appears useful in assessment of glioma cell proliferation. These results indicate that two-dimensional polyacrylamide gel electrophoresis can contribute to the understanding of the biological features of glioma cells.
Honda
C
C
Tabuchi
K
K
Nishimoto
A
A
D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D005910:Glioma / Q000032:analysis* / Q000276:immunology; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen; D051381:Rats; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000032:analysis / Q000276:immunology; D014867:Water
D000951:Antigens, Neoplasm; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D014867:Water