The author analyzed water-soluble proteins of cultured human and rat glioma cells obtained by freezing and thawing cells suspended in distilled water. The author used O'Farrell's two dimensional polyacrylamide gel electrophoresis (NEPHGE method) with slight modification to analyze differences in the protein map, and Manabe's microscale two dimensional electrophoresis without denaturing agents for Western blotting to detect proliferating cell nuclear antigen (PCNA/cyclin). By O'Farrell's method and the silver staining technique, at least 200 different polypeptides were clearly identified in each cell line. Cytoskeletal proteins such as actin were constantly 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. It is supposed that these differences arise from the cell-biological differences between the glioma cell lines. Marked differences in the portion map were also seen between proliferating glioma cells and glioma cells treated with forskolin (an adenylate cyclase activator), which can induce morphological and biochemical differentiation of glioma cells. Some proteins increased in amount after the treatment with forskolin, suggesting that these proteins are related to the differentiation of glioma cells. Some other proteins which were prominent in the proliferating cells diminished in cells cultured 24 hours in medium without calf serum to suppress cell growth. PCNA (proliferating cell nuclear antigen), an acidic nuclear protein which appears solely in late G(1)-S phase and is believed to be closely related to cell proliferation, was revealed by Western blotting and indirect immunostaining. The quantitative investigation of the PCNA spot on the protein map may be useful in assessing the proliferating activity of glioma cells. These results indicate that two dimensional polyacryamide gel electrophoresis is useful for the clarification and understanding of the biological features of glioma cells.
water soluble protein
two dimensional electrophoresis
proliferating cell nuclear antigen (PCNA)