Abstract
U-251 MG, a permanent cell line derived from human glioblastoma multiforme with the capacity to maintain glial fibrillary acidic protein (GFAP) production over repeated in vitro passages, was evaluated for the expression of three neuron-associated proteins (Class III β-tubulin, MAP2, and tau) in three different in vitro systems: as free-floating suspensions, on coverslips, and on a gelatin foam (Gelfoam) matrix. Cells grown under the three in vitro conditions were analyzed by immunoblotting techniques, whereas immunohistochemical analyses were performed on cells grown on Gelfoam. By immunohistochemistry, cells were positive for Class III β-tubulin isotype, a neuronassociated β-tubulin, for microtubule-associated protein 2 (MAPs), but not for tau. Immunoblotting studies confirmed the presence of Class III β-tubulin in extracts of cells grown under the three in vitro conditions. MAP2 and tau were clearly evident only in cell extracts grown in Gelfoam cultures. GFAP expression was observed in all three in vitro conditions by immunoblotting and also in foam matrix cultures by immunohistochemistry. In matrix cultures, Class III β-tubulin- and GFAP-positive cells were found immediately adjacent to each other, but coexpression of these proteins was not observed, and the cells were morphologically indistinguishable. Our findings confirm the heterogeneity of malignant gliomas in vitro, and the implications of these observations require further study.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bigner, D. D., Bigner S. H., Pontén J., Westermark B., Mahaley Jr. M. S., Ruoslahti E., Herschman H., Eng L. F., and Wikstrand C. J. (1981a) Heterogeneity of genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas.J. Neuropathol. Exp. Neurol. 40, 201–229.
Bigner S. H., Bullard D. E., Pegram C. N., Wikstrand C. J. and Bigner D. D. (1981b) Relationship of in vitro morphologic and growth characteristics of established human glioma-derived cell lines to their tumorigenicity in athymic nude mice.J. Neuropathol. Exp. Neurol. 40, 390–409.
Binder L. I., Frankfurter A., Kim H., Caceres A., Payne M. R., and Rebhun L. I. (1984) Heterogeneity of microtubule-associated protein 2 during rat brain development.Proc. Natl. Acad. Sci. USA 81, 5613–5617.
Binder L. I., Frankfurter A., and Rebhun L. I. (1985) The distribtion of tau in the mammalian central nervous system.J. Cell Biol. 101, 1371–1378.
Bissell M. G., Rubinstein L. J., Bignami A., and Herman M. M. (1974) Characteristics of the rat C-6 glioma maintained in organ culture systems. Prodution of glial fibrillary acidic protein in the absence of gliofibrillogenesis.Brain Res. 82, 77–89.
Bissell M. G., Eng L. F., Herman M. M., Bensch K. G., and Miles L. E. M. (1975) Quantitative increase in neuroglia-specific GFA protein in rat C-6 glioma cells in vitro.Nature 255, 633, 634.
Burgoyne R. D. (1986) Microtubule proteins in neuronal differentiation.Comp. Biochem. Physiol. 83B, 1–8.
Caccamo D. V., Katsetos C. D. Frankfurter A., Collins V. P., VandenBerg S. R., and Herman M. M. (1989a). An immunohistochemical characterization of the primitive and maturing neuroepithelial component in the OTT-6050 transplantable mouse teratoma.Neuropathol. Appl. Neurobiol. 15, 389–405.
Caccamo D., Katsetos C. D., Herman M. M., Frankfurter A., Collins V. P., and Rubinstein, L. J. (1989b) Immunohistochemistry of a spontaneous murine ovarian teratoma with neuroepithelial differentiation.Lab. Invest. 60, 390–398.
Caccamo D. V., Herman M. M., and Rubinstein L. J. (1989c) An immunohistochemical study of the primitive and maturing elements of human cerebral medulloepitheliomas.Acta Neuropathol. 79, 248–254.
Collins V. P. (1983) Cultured human glial and glioma cells.Int. Rev. Exp. Pathol. 24, 135–202.
Couchie D., Charriere-Bertrand C., and Nuñez J. (1988) Expression of the mRNA for tau proteins during brain development and in cultured neurons and astroglial cells.J. Neurochem. 50, 1894–1899.
Frankfurter A., Binder L. I., and Rebhun L. I. (1986) Limited tissue distribution of a novel β-tubulin isoform.J. Cell Biol. 103, 273a.
Garner C. C. and Matus A. (1988) Different forms of microtubule-associated protein 2 are encoded by separate mRNA transcripts.J. Cell Biol. 106, 779–783.
Garner C. C., Brugg B., and Matus A. (1988) A 70-kilodalton microtubule-associated protein (MAP2c), related to MAP2.J. Neurochem. 50, 609–615.
Geisert Jr. E. E., Johnson H. G., and Binder L. I. (1990) Expression of microtubule-associated protein 2 by reactive astrocytes.Proc. Natl. Acad. Sci. USA 87, 3967–3971.
Katsetos C. D., Herman M. M., Frankfurter A., Gass P., Collins V. P., Walker C. C., Rosemberg S., Barnard R. O., and Rubinstein L. J. (1989) Cerebellar desmoplastic medulloblastomas—a further immunohistochemical characterization of the reticulin-free pale islands.Arch. Pathol. Lab. Med. 113, 1019–1029.
Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685.
Lee M. K., Tuttle J. B., Rebhun L. I., Cleveland D. W., and Frankfurter A. (1990) The expression and post-translational modification of a neuron-specific β-tubulin isotype during chick embryogenesis.Cell Motil. Cytoskeleton 17, 118–132.
Matus A. (1988) Microtubule-associated proteins: their potential role in determining neuronal morphology.Annu. Rev. Neurosci. 11, 29–44.
Moody S. A., Quigg M. S., and Frankfurter A. (1989) Development of the peripheral trigeminal system in the chick revealed by an isotype-specific anti-β-tubulin monoclonal antibody.J. Comp. Neurol. 279, 567–580.
Mörk S. J. and Laerum O. D. (1980) DNA distribution in human gliomas in vivo and in vitro.Acta Pathol. Micriobiol. Scand. 274 (Suppl.), 403–406.
Nakano I., Iwatsubo T., Otsuba N., Kamei M., Matsumura K., and Mannen T. (1992) Paired helical filaments in astrocytes: Electron microscopy and immunohistochemistry in a case of atypical Alzheimer’s disease.Acta Neuropathol. 83, 228–232.
Olmsted J. B. (1986) Microtubule-associated proteins.Annu. Rev. Cell Biol. 2, 421–457.
Paetau A. and Virtanen I. (1986) Cytoskeletal properties and endogeneous degradation of glial fibrillary acidic protein and vimentin in cultured human glioma cells.Acta Neuropathol. 69, 73–80.
Papasozomenos S. Ch. (1989) Tau protein immunoreactivity in dementia of the Alzheimer Type. I. Morphology, evolution, distribution, and pathogenetic implications.Lab. Invest. 60, 123–137.
Papasozomenos S. Ch. and Binder L. I. (1986) Microtubule-associated protein 2 (MAP2) is present in astrocytes of the optic nerve but absent from astrocytes of the optic tract.J. Neurosci. 6, 1748–1755.
Papasozomenos S. Ch., Binder L. I., Bender P., and Payne M. R. (1985) Microtubule-associated protein 2 within axons of spinal motor neurons: Associations with microtubules and neurofilaments in normal and β,β′-iminodipropionotrile-treated axons.J. Cell Biol. 100, 74–85.
Pontén J. and Westermark B. (1978) Properties of human malignant glioma cells in vitro.Med. Biol. 56, 184–193.
Rubinstein L. J., Herman M. M., and VandenBerg S. R. (1987) Differentiation and anaplasia in central neuroepithelial tumors.Prog. Exp. Tumor Res. 27, 32–48.
Shapiro J. R., Yung W. A., and Shapiro W. R. (1981) Isolation, karyotype, and clonal growth of heterogeneous subpopulations of human malignant gliomas.Cancer Res. 41, 2349–2359.
Shin R.-W., Iwaki T., Kitamoto T., and Tateishi J. (1991) Hydrated autoclave pretreatment enhances tau immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissue.Lab. Invest. 64, 693–702.
Sternberger L. A., Hardy Jr. P. H., Cuculis J. J., and Meyer H. G. (1970) The unlabeled antibody enzyme method of immunohistochemistry.J. Histochem. Cytochem. 18, 315–333.
Sullivan K. F. (1988) Structure and utilization of tubulin isotypes.Annu. Rev. Cell Biol. 4, 687–716.
Trimmer P. A., Frankfurter A., and Rebhun L. I. (1986) Immunocytochemical staining of neural cultures with a novel b-tubulin monoclonal antibody.J. Cell Biol. 103, 273a.
Tucker R. P., Viereck L. I., Hemmings B. A., and Matus A. I. (1988) The sequential appearance of low- and high-molecular-weight forms of MAP2 in the developing cerebellum.J. Neurosci. 8, 4503–4512.
Vaughn J. E. and Pease D. C. (1970) Electron microscopy studies of Wallerian degeneration in rat optic nerves. II. Astrocytes, oligodendrocytes and adventitial cells.J. Comp. Neurol. 140, 207–226.
Yamada T., McGeer P. L., and McGeer E. G. (1992) Appearance of paired nucleated, tau-positive glia in patients with progressive supranuclear palsy.Neurosci. Lett. 135, 99–102.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lopes, M.B.S., Frankfurter, A., Zientek, G.M. et al. The presence of neuron-associated microtubule proteins in the human U-251 MG cell line. Molecular and Chemical Neuropathology 17, 273–287 (1992). https://doi.org/10.1007/BF03160016
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF03160016