CLASS-III BETA-TUBULIN ISOTYPE (BETA-III) IN THE ADRENAL-MEDULLA - III - DIFFERENTIAL EXPRESSION OF NEURONAL AND GLIAL ANTIGENS IDENTIFIES 2 DISTINCT POPULATIONS OF NEURONAL AND GLIAL-LIKE (SUSTENTACULAR) CELLS IN THE PC12 RAT PHEOCHROMOCYTOMA CELL-LINE MAINTAINED IN A GELFOAM MATRIX SYSTEM

Background: The rat PC12 pheochromocytoma cell line provides an establ ished system for the study of neuronal differentiation. To our knowled ge, glial differentiation has not been reported in this cell line. Met hods: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens [class III beta-tubulin is otype (beta III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components], and synaptophys in in comparison with the glial fibrillary acidic protein (GFAP) and S -100 protein in the PC12 cell line, In three different experiments, PC 12 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 explan ts ranging from 2 to 32 days-in vitro, which were fixed in either Boui n's solution, 70% ethanol, or 10% neutral-buffered formalin and embedd ed in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial ma rkers, Additional immunoblot experiments using anti-GFAP and anti-beta III monoclonal antibodies in cell suspensions and homogenates from PC 12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins. Results: beta III and M AP2 were demonstrated by immunohistochemistry and immunoblotting of PC 12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP, Intense filamentous and granular beta II I staining of PC12 cells was observed in dcAMP-treated cultures concom itant with neuronal morphologic alterations (neuritogenesis and gangli onic phenotype), In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic dev elopment, The neuronal phenotype of PC12 cells was confirmed by staini ng for MAP2, tau, and NF proteins, as well as for synaptophysin, The p resence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed b y immunoblotting, Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or ne uronal cells, were demonstrated in Gelfoam explants at 5-30 days in vi tro, In 30-day-old cultures treated with dcAMP, there was strong filam entous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells. GFAP staining was corroborated by im munoblotting of explants maintained under identical conditions in vitr o, In contrast, immunoblots performed on homogenates from PC12 suspens ion and monolayer cultures were GFAP-negative. Conclusions: Neuronal a nd glial-like, presumed sustentacular, phenotypes were demonstrated in PC12 cells grown in Gelfoam matrices with and without treatment with dcAMP for up to 34 days. To our knowledge, the occurrence of glial dif ferentiation in the PC12 line is a hitherto unreported finding, Adult rat medullary sustentacular cells are known to express S-100 and GFA p roteins (Suzuki and Kachi, Kaibogaku Zasshi-J Anat 70(2): 130-139, 199 5), and the organ culture system employed in our study may well have f avored this direction of differentiation. (C) 1998 Wiley-Liss, Inc.