Neuron-associated class III ?-tubulin, tau, and MAP2 in the D-283 Med cell line and in primary explants of human medulloblastoma

Abstract

The D283 Med human medulloblastoma cell line and primary explants of five surgically excised medulloblastomas were cultured using a three-dimensional Gelfoam matrix system. The cultures were evaluated immunohistochemically for a series of antigenic determinants associated with neuronal or glial differentiation. Focal immunolocalization of class III beta-tubulin, microtubule-associated protein 2 (MAP2), and to a lesser degree tau, was demonstrated in all cultures. Class III beta-tubulin isotype, MAP2, and tau protein were also detected by immunoblot in Gelfoam matrix cultures, monolayer cultures, and suspension cultures of D283 Med cells. Staining for neurofilament protein epitopes was highly variable, even among different cultures derived from the same original tumour, but time-dependent changes in neurofilament protein, which may have reflected neuronal differentiation, were not consistently shown. Widespread gamma-enolase and focal synaptophysin reactivities were visualized in all cultures, but no S-antigen staining was detected. Leu 7 labelling was variably present in half of the cultures of D283 Med cells, but was more abundant in explants derived from four of the five original tumours. Vimentin was consistently found in D283 Med cultures at all time points. No immunoreactivity for glial fibrillary acidic protein was detected in the D283 Med cell line. Conversely, staining for this protein was demonstrated in scattered astrocytic cells in the surgical specimens of all five medulloblastomas. Concomitant with increased time in culture, three of the primary tumours displayed increased numbers of glial fibrillary acidic protein-positive cells when cultured in the Gelfoam system, but the other two tumours had a minimal astrocytic component.(ABSTRACT TRUNCATED AT 250 WORDS)

Class III β‐tubulin in human development and cancer

“…Based on differential patterns of localization of ␤III and PCNA/cyclin, four neuroblastic tumor phenotypes (Nb1-Nb4) have been defined reflecting gradations of neuronal differentiation in medulloblastomas [Katsetos and Burger, 1994;Katsetos et al, 1995Katsetos et al, , 2003. Moreover, ␤III is expressed in primary medulloblastoma explants and in the human medulloblastoma cell line (D283-Med) maintained in an organ culture system [Vinores et al, 1994].…”

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

“…Evidence for the characterization and specificity of most primary antibodies is presented in previous studies as follows: mouse mAb TuJ1 against ␤ III (produced by AF) Katsetos et al, 1989;Lee et al, 1990;Gass et al, 1990;Vinores et al, 1994), mouse mAb AP18 against MAP2 (produced by AF) Katsetos et al, 1989;Gass et al, 1990), mouse mAb Tau-1 against tau protein (produced by AF) (Vinores et al, 1994), mouse mAb TNFP1A3 against bovine neurofilament protein (presumed phosphoepitope of the heavy neurofilament subunit) (gift of V.P. Collins) Katsetos et al, 1989), mouse mAb SY38 against synaptophysin (Boehringer-Mannheim, Indianapolis, IN) (Katsetos et al, , 1991, a mouse mAb against GFAP (gift of Dr. V.P.…”

“…Thirty-two-day explants of the human medulloblastoma cell line D283 Med (Vinores et al, 1994) and the human glioblastoma cell line U-251 MG (Lopes et al, 1992) were examined simultaneously for the presence of GFAP. The methods for cell homogenate preparation, gel electrophoresis and immunoblotting are detailed in Lopes et al (1992).…”

“…A three-dimensional gelatin foam (Gelfoam) matrix has been established as a culture system for studying differentiation in neural tumors (Rubinstein et al, 1973;Halks-Miller et al, 1981;Herman et al, 1989;Gass et al, 1990;Vinores et al, 1994). We elected to use the Gelfoam matrix system since it facilitates differentiation of neural cells by virtue of maintaining cell-tocell interactions analogous to in vivo growth (Rubinstein and Herman, 1975).…”

Preterm Delivery Disrupts the Developmental Program of the Cerebellum

“…Therefore, we investigated whether premature birth could lead to a downregulation of the Shh pathway, leading to a thinning of the EGL layer. The expression of doublecortin was used as a marker for cells that had exited the cell cycle and commenced migration and β-III tubulin was used as a marker for neuronal differentiation [27]. Calbindin and ITPR were used as specific markers for purkinje cell differentiation [28]–[30] and GFAP as a marker for bergmann glia differentiation [31].…”