ABSENCE OF NEURON-ASSOCIATED MICROTUBULE PROTEINS IN THE RAT C-6 GLIOMA CELL-LINE - A COMPARATIVE IMMUNOBLOT AND IMMUNOHISTOCHEMICAL STUDY

Three neuron-associated microtubule proteins, Class III beta-tubulin i sotype, MAP-2, and tau, were evaluated in a comparative immunoblot and immunohistochemical study of the rat C-6 glioma cell line maintained for up to 3 1 days in vitro. Western blots on whole SDS extracts of ce lls grown: (i) as monolayers on plastic dishes (for 13 and 16 days); ( ii) as monolayers on poly-D-lysine coated glass coverslips (for 3, 7, and 11 days); and (iii) as explants on Gelfoam matrices (for 10, 30, a nd 31 days) were probed with monoclonal antibodies (MoAb) specific for the above-mentioned microtubule proteins. For these and all other mar kers employed, immunoperoxidase histochemistry was performed only on t he matrix cultures. The immunoblot experiments demonstrated that the C lass III beta-tubulin isotype, MAP2, and tau were not expressed by the C-6 cell line in any of the culture conditions, nor were they found b y immunohistochemistry. In contrast, explants from all culture conditi ons were positive for glial fibrillary acidic (GFA) protein and for a universal anti-beta-tubulin isotype MoAb by immunoblotting, as well as by immunohistochemistry in Gelfoam matrix cultures maintained in an o rgan culture system. Both sets of experiments indicate that these mark ers are not altered under three different conditions of growth over a one-month period in vitro. The expression of GFA protein and the absen ce of detectable levels of Class III beta-tubulin, MAP2, and tau are i n keeping with the astrocytic phenotype of the C-6 cell line.