Class III β-Tubulin isotype (β III) in the adrenal medulla: III. Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial-like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a gelfoam matrix system

Abstract

Background: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation. To our knowledge, glial differentiation has not been reported in this cell line.Methods: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens [class III ␤-tubulin isotype (␤ III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components], and synaptophysin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line. In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP. Immunohistochemistry was performed on explants ranging from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers. Additional immunoblot experiments using anti-GFAP and anti-␤ III monoclonal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins.

Distribution of the class II β‐tubulin in developmental and adult rat tissues

“…These observations imply that the class II isotype may play a transient role in later stages of neuronal development, such as those of post-mitotic neurons. Additionally, positive immunoreactivity in the extended processes of NGF-stimulated PC 12 cells also suggests that this isotype is important for neurite growth, similar to the class III isotype shown in the previous reports [Joshi and Cleaveland, 1989;Katsetos et al, 1998b]. Cells of mesoderm-origin, such as fibroblasts, myoblasts, and chondrocytes, showed a strong immunoreactivity in developmental non-neuronal tissues, and show a diminished immunoreactivity by varying degrees in adulthood.…”

Multipotent Glia-Like Stem Cells Mediate Stress Adaptation

“…The glia‐like properties of the nestin–GFP positive stem cells and their neural differentiation potential might also explain the frequent observation of pheochromocytomas with sustentacular properties . This dual differentiation was also observed in the rat pheochromocytoma cell line PC12 although it is supposed to be monoclonal , suggesting that PC12 cells might be derived from a multipotent adrenomedullary precursor cell with properties characterized here.…”

Simultaneous Suppression of cdc2 and cdk2 Activities Induces Neuronal Differentiation of PC12 Cells

“…At days 0, 1, 3, 5, 7, and 10, cells were lysed and the expression of cdc2 and cdk2 was evaluated by immunoblotting analysis using specific antibodies. Changes in MAP2 expression were also examined to confirm the neuronal differentiation of PC12 cells induced by NGF (27,34). As shown in Fig.…”

“…Both of these agents have been previously demonstrated to suppress cdc2/cdk2 activity (23, 24, 35). In addition, changes in MAP2 expression were also examined to confirm the neuronal differentiation of PC12 cells by NGF (27,34). A control group, treated with 0.1% dimethyl sulfoxide alone did not show significant neurite outgrowth and expression of MAP2 was much lower than in cells treated with NGF (50 ng/ml) for 3 days (Fig.…”