Aberrant localization of the neuronal class III beta-tubulin in astrocytomas.

BACKGROUND: The class III beta-tubulin isotype (betaIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of betaIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. OBJECTIVE: To test the generality of this observation, we investigated the immunoreactivity profile of betaIII in astrocytomas. DESIGN: Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-betaIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. RESULTS: The betaIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%-47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%-21%) (P <.0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%-0.5%) (P <.0001 vs high-grade astrocytomas; P <.01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between betaIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for betaIII (betaIII, P <.006; Ki-67, P <.0001). There was co-localization of betaIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. CONCLUSIONS: In the context of astrocytic gliomas, betaIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of betaIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, betaIII expression is not neuron specific, calling for a cautious interpretation of betaIII-positive phenotypes in brain tumors.

Differential distribution of the neuron-associated class III beta-tubulin in neuroendocrine lung tumors.

OBJECTIVE: To study the immunoreactivity profile of the neuron-associated class III beta-tubulin isotype (beta III) in epithelial lung tumors. DESIGN: One hundred four formalin-fixed, paraffin-embedded primary and metastatic lung cancer specimens were immunostained with an anti-beta III mouse monoclonal antibody (TuJ1) and an anti-beta III affinity-purified rabbit antiserum. Paraffin sections from fetal, infantile, and adult nonneoplastic lung tissues were also examined. RESULTS: In the fetal airway epithelium, beta III staining is detected transiently in rare Kulchitsky-like cells from lung tissues corresponding to the pseudoglandular and canalicular but not the saccular or alveolar stages of development. beta III is absent in healthy, hyperplastic, metaplastic, and dysplastic airway epithelium of the adult lung. In contrast, beta III is highly expressed in small cell lung cancer, large cell neuroendocrine carcinoma, and in some non-small cell lung cancers, particularly adenocarcinomas. There is no correlation between expression of beta III and generic neuroendocrine markers, such as chromogranin A and/or synaptophysin, in pulmonary adenocarcinomas. Also, focal beta III staining is present in primary and metastatic adenocarcinomas (to the lung) originating in the colon, prostate, and ovary. beta III is expressed to a much lesser extent in atypical carcinoids and is rarely detectable in typical carcinoids and squamous cell carcinomas of the lung. The distribution of beta III in small cell lung cancer and adenocarcinoma metastases to regional lymph nodes and brain approaches 100% of tumor cells, which is substantially greater than in the primary tumors. CONCLUSIONS: In the context of neuroendocrine lung tumors, beta III immunoreactivity is a molecular signature of high-grade malignant neoplasms (small cell lung cancer and large cell neuroendocrine carcinoma) although its importance in atypical carcinoids must be evaluated further. In addition, beta III may be a useful diagnostic marker in distinguishing between small cell lung cancers and certain non-small cell lung cancers (poorly differentiated squamous cell carcinomas), especially in small biopsy specimens. To our knowledge, beta III is the only tumor biomarker that exhibits a substantially more widespread distribution in poorly differentiated than in better differentiated pulmonary neuroendocrine tumors. However, the significance of beta III phenotypes in non-small cell lung cancer, particularly adenocarcinoma, with respect to neuroendocrine differentiation and prognostic value, requires further evaluation.

Posttranslational modifications of the C-terminus of alpha-tubulin in adult rat brain: alpha 4 is glutamylated at two residues.

In adult mammalian brain, the C-terminus of alpha-tubulin exhibits a high degree of polymorphism due to a combination of four covalent posttranslational modifications: glutamylation, tyrosination, detyrosination, and removal of the penultimate glutamate residue (C-terminal deglutamylation). Glutamylation is the most abundant. To characterize the glutamylation of alpha-tubulin and its relationship with the other modifications, we developed a chromatographic procedure for purifying alpha-tubulin C-terminal peptides. The purified peptides were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. In this report, we provide a complete description of the glutamylation of tyrosinated, detyrosinated, and C-terminal deglutamylated isoforms of both alpha-tubulin isotypes (alpha1/2 and alpha4) expressed in adult rat brain. In particular, we describe for the first time the glutamylation of alpha4. More than 90% of the alpha-tubulin is glutamylated, and more than 75% of it is nontyrosinated. alpha4 is more extensively glutamylated than alpha1/2, containing as many as 11 posttranslationally added glutamate residues. The most abundant alpha4 isoform is nontyrosinated, containing five posttranslationally added glutamates, whereas the most abundant alpha1/2 isoforms are nontyrosinated, with only one or two posttranslationally added glutamates. In contrast to alpha1/2, alpha4 is glutamylated at two separate residues (Glu-443 and Glu-445) in the sequence 431DYEEVGIDSYEDEDEGEE448. This is the first evidence that glutamylation can occur on two different residues in the same mammalian tubulin isotype.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: I. Localization in the developing human adrenal medulla.

BACKGROUND: The class III beta-tubulin isotype (beta III) is present in neurons of the central and peripheral nervous systems at the earliest stages of morphological differentiation (Easter et al., J Neurosci 13:285-299, 1993; Katsetos et al., J Neuropathol Exp Neurol 52:655-666, 1993). The localization of this protein by immunohistochemistry in the different cell types of the developing human adrenal medulla is described. METHODS: A mouse monoclonal antibody, TuJ1, was used to localize beta III in formalin-fixed, paraffin-embedded sections from 18 human fetal and adult adrenal glands. Tissue sections were also studied with rabbit antisera recognizing either S-100 protein or glial fibrillary acidic protein (GFAP). RESULTS: In the developing human adrenal medulla, beta III immunoreactivity was maximal in migrating sympathoadrenal neuroblasts/immature neurons through the end of the second trimester. Clusters of beta III-positive migrating cells, focally forming Homer Wright rosettes, could be identified in a gradient of adrenocortical invasion, i.e., through the permanent cortex and within sinusoids of the fetal cortex en route to the medulla. Outside the adrenal gland, strong beta III staining was observed in peripheral nerve bundles, sympathetic ganglia, and paraganglia at various developmental stages. In adrenal glands from 23 weeks of gestation on, and throughout adult life, all ganglion cells were beta III immunoreactive. In contrast, not all chromaffin cells exhibited beta III staining, but when present, the staining was finely granular. Sustentacular and satellite cells, adrenocortical cells and other mesenchymal elements were betaIII-negative. In sections of fetal and adult adrenal glands, S-100 protein had a sustentacular localization. No GFAP staining was present in sustentacular cells from either fetal or adult adrenals. CONCLUSIONS: In the developing human adrenal medulla, there is a peak of beta III expression during the active wave of migration of sympathetic neuroblasts. In the mature medulla, beta III is invariably present in adrenergic neurons. However, not all chromaffin-like cells express beta III, suggesting that the presence or absence of this protein identifies two subpopulations of chromaffin cells.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: II. Localization in primary human pheochromocytomas.

BACKGROUND: The Class III beta-tubulin isotype (beta III) is expressed specifically in central and peripheral nervous system neurons at various stages of neuronal differentiation. We have shown previously that beta III is expressed in a differentiation-dependent manner in human neuroblastomas arising in the adrenal medulla and sympathetic chains (Katsetos et al., Clin Neuropathol 13:241-255, 1994). The neuronal distribution of beta III in the developing and mature human adrenal medullae is detailed in the companion article (Katsetos et al., 1998A). METHODS: We have compared the localization of the neuronal beta III to S-100 protein, a sustentacular cell marker, in 14 formalin-fixed, paraffin-embedded primary human pheochromocytomas of the adrenal medulla and 14 adrenocortical tumors (adenomas and carcinomas). RESULTS: In pheochromocytomas, beta III staining was present in all tumors, but the number of stained cells varied in the two neural neoplastic phenotypes. Although the majority of chromaffin-like cells were beta III-positive, there was a lack of beta III in one-third of the tumor cells. Compared to chromaffin-like phenotypes, neuronal (ganglion-like cells) were invariably beta III-positive. Stromal sustentacular cells, stromal fibroblasts, and tumor blood vessels were beta III-negative. Sustentacular cells in pheochromocytomas were S-100 protein-positive, but beta III-negative. Primary adrenocortical tumors were beta III-negative with the exception of rare beta III-positive cells demonstrated in one case. CONCLUSIONS: The distribution of beta III in human pheochromocytomas of the adrenal gland is differentiation-dependent, closely recapitulating chromaffin cell and neuronal phenotypes of the normal adrenal medulla. Our findings indicate that beta III may be used as one of the adjuvant neural markers in the differential diagnosis of adrenal tumors, i.e., pheochromocytoma versus adrenocortical carcinoma. The occurrence of rare beta III-positive cells in cortical carcinomas is exceptional and probably represents the acquisition of a divergent neuroendocrine phenotype. The significance of the latter is unclear, although it may constitute a marker for malignancy.

Class III beta-tubulin isotype (beta 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.

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 beta-tubulin isotype (beta 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-beta 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. RESULTS: Beta III and MAP2 were demonstrated by immunohistochemistry and immunoblotting of PC12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP. Intense filamentous and granular beta III staining of PC12 cells was observed in dcAMP-treated cultures concomitant with neuronal morphologic alterations (neuritogenesis and ganglionic phenotype). In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic development. The neuronal phenotype of PC12 cells was confirmed by staining for MAP2, tau, and NF proteins, as well as for synaptophysin. The presence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed by immunoblotting. Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or neuronal cells, were demonstrated in Gelfoam explants at 5-30 days in vitro. In 30-day-old cultures treated with dcAMP, there was strong filamentous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells. GFAP staining was corroborated by immunoblotting of explants maintained under identical conditions in vitro. In contrast, immunoblots performed on homogenates from PC12 suspension and monolayer cultures were GFAP-negative. CONCLUSIONS: Neuronal and 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 differentiation in the PC12 line is a hitherto unreported finding. Adult rat medullary sustentacular cells are known to express S-100 and GFA proteins (Suzuki and Kachi, Kaibogaku Zasshi-Anat 70(2): 130-139, 1995), and the organ culture system employed in our study may well have favored this direction of differentiation.

Energetics of vinca alkaloid interactions with tubulin isotypes: implications for drug efficacy and toxicity.

A number of vinca alkaloids, including vincristine, vinblastine, and vinorelbine, are currently used in cancer chemotherapy. These three vinca alkaloids interact differently with a range of solid and hematologic tumors. To test the possibility that the tubulin isotype composition is an important determinant in antineoplastic efficacy, we determined thermodynamic parameters for vinca alkaloid interactions with purified beta-tubulin isotypes, alphabetaII or alphabetaIII, as well as mixtures of alphabetaII and alphabetaIII, alphabetaII and alphabetaI&IV, or alphabetaIII and alphabetaI&IV (referred to as isotype-depleted tubulin) by quantitative sedimentation velocity. Vincristine-, vinblastine-, or vinorelbine-induced isotype self-association was studied at 25 degrees C in 10 mM Pipes, pH 6.9, 1 mM MgSO4, and 2 mM EGTA in the presence of 50 microM GTP or GDP. For all three drugs, we observed no significant differences in overall affinities, K1K2, or in GDP enhancement of purified isotypes compared to unfractionated tubulin, suggesting that differential antitumor efficacy observed clinically for these vinca alkaloids is not determined by tissue isotype composition. Small, but significant differences in the individual binding parameters, K1 and K2, are found in the vincristine data. In the presence of vincristine and GTP, K1, the affinity of drug for tubulin heterodimers, tends to be larger for purified alphabetaII- or alphabetaIII-tubulin compared to unfractionated tubulin. Furthermore, the apparent dimerization constant, K2app, at physiologically significant drug concentrations is larger for these purified isotypes. When alphabetaII- and alphabetaIII-tubulin are combined, the cooperativity between drug binding and spiral formation approaches that of unfractionated PC-tubulin. These differences are not observed in the presence of vinblastine or vinorelbine. The differences found with vincristine may be implicated in the dose-limiting neurotoxicity found with this drug, but not found with vinblastine or vinorelbine.

Developmental expression of a neuron-specific beta-tubulin in frog (Xenopus laevis): a marker for growing axons during the embryonic period.

In mammals, there are seven classes of beta-tubulin genes, one of which, class III, is neuron specific. Using class-specific monoclonal antibodies, class III beta-tubulin protein could not be detected in frog embryos or in adults with either Western blotting or immunohistochemical techniques. In contrast, the class II beta-tubulin protein, which is predominant in mammalian brain but is also expressed in other tissues, is expressed only in neurons in frog embryos. Protein was detected only in neurons from late stages of neural tube closure through premetamorphic stages. At stages 21-28, the pioneering axons of Rohon-Beard, commissural, primary motor, and trigeminal ganglion neurons were distinctly stained in the axon scaffolds that they formed in the embryonic brain and the peripheral mesenchyme. Nonneuronal cells, both outside the nervous system and within it (e.g., radial glia, Müller glia, roof plate, and floor plate cells) were not immunoreactive. Throughout swimming and premetamorphic stages, neuronal cells in all brain regions became immunoreactive as they differentiated and extended axons. Whereas many embryonic neurons became postmitotic during gastrulation stages, neurons expressed detectable levels of class II beta-tubulin protein only beginning at the onset of overt axon outgrowth. These observations demonstrate that the neuron-specific beta-tubulin in frog is a different gene from that in mammals, and its protein product is detectable at the time of axonogenesis rather than neurogenesis.

Binding of vinblastine to phosphocellulose-purified and alpha beta-class III tubulin: the role of nucleotides and beta-tubulin isotypes.

Vinblastine is an antimitotic drug that inhibits microtubule assembly and induces the self-association of tubulin into coiled spiral aggregates. Previous quantitative binding and sedimentation velocity results have been interpreted by a mechanism involving isodesmic ligand-mediated plus ligand-facilitated self-association [Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214-6222, 6222-6228]. In this study, the vinblastine-induced self-association of porcine brain tubulin has been compared in the presence of 50 microM GDP or 50 microM GTP to investigate the role of nucleotides. Experiments at 1-4 microM tubulin in 10 mM Pipes, 1 mM MgSO4, 2 mM EGTA (pH 6.9), and varying concentrations of vinblastine (0.05-70 microM) demonstrate that GDP enhances self-association by 2-4-fold over GTP. In the presence of GDP or GTP, sedimentation velocity data can be best fit by either an indefinite ligand-mediated model or an indefinite ligand-mediated plus ligand-facilitated model. The association constant, K2, for the vinblastine-tubulin complex binding to a polymer is larger when GDP is present, while the association constant, K1, for the binding of vinblastine to tubulin heterodimers is identical in the presence of either nucleotide. The enhancement of K2 by GDP is confirmed by micropartition binding experiments with [3H]vinblastine. The fitting of sedimentation velocity and binding studies gives parameters for the interaction of vinblastine with GTP-tubulin that are identical, within error, to the previous results of Na and Timasheff. van't Hoff analysis of multiple temperature data reveals that this enhancement in the presence of GDP is due to a change in the enthalpy of self-association. Additional results suggest that the interaction of vinblastine with tubulin is identical for all beta-isotypes. Sedimentation velocity experiments in the presence of GDP or GTP show that the vinblastine-induced association of affinity-purified alpha,beta-class III tubulin is identical to that of unfractionated tubulin, although there is a difference in the abilities of unfractionated tubulin and alpha,beta III-tubulin to associate into taxol-stabilized microtubules.

Class III beta-tubulin in human retinal pigment epithelial cells in culture and in epiretinal membranes.

The class III beta-tubulin isotype (beta III) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in 1 day-old primary cultures; however, beta III is present in RPE cells in 5-day primary cultures and in passaged RPE cells grown in monolayer cultures as determined by immunohistochemistry and Western blotting. beta III-positivity in cultured RPE cells is not affected by cell density or hydroxyurea- or retinoic-acid-mediated growth inhibition, but only a few cells weakly express beta III in cyclohexamide-treated cultures and RPE cells maintained in serum-free medium fail to produce beta III. When monolayer-cultured RPE cells grown in normal, serum-containing medium, are transferred to irradiated bovine vitreous, beta III is undetectable in most cells. Cultured RPE cells coexpress beta III with keratin and cellular retinaldehyde-binding protein (both RPE cell markers), but not with glial fibrillary acidic protein. Some cultured RPE cells also express neuron-specific (gamma) enolase, which is neuron-associated but not neuron-specific, and occasional cells in confluent or super-confluent cultures contain the 200-kDa neurofilament protein. Retinal glia, fibroblasts, endothelial cells, and smooth muscle cells do not express beta III under the same culture conditions. We have detected beta III in 45 of 56 epiretinal membranes, frequently in cells with a bipolar or dedifferentiated morphology, where its expression coincides with other RPE cell-associated antigens. Cells with morphological features resembling normal RPE cells in epiretinal membranes are usually negative for beta III, but RPE cells appearing to be in the early stages of dedifferentiation express the isotype weakly. Electron microscopic immunocytochemistry localizes beta III to microtubules, ribosomes and cytoplasm. beta III may be a useful marker for recognizing the fraction of RPE cells in epiretinal membranes that are no longer identifiable by morphological criteria or other RPE cell markers. These findings demonstrate that mature human RPE cells have the capacity to express a neuron-associated gene in response to conditions that promote dedifferentiation.

A cytomorphological scheme of differentiating neuronal phenotypes in cerebellar medulloblastomas based on immunolocalization of class III beta-tubulin isotype (beta III) and proliferating cell nuclear antigen (PCNA)/cyclin.

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III) to that of the proliferating cell nuclear antigen (PCNA)/cyclin in 46 cerebellar neuroblastic tumors (medulloblastomas). Both class III beta-tubulin (beta III) and PCNA/cyclin reactivities were present in all tumors, but the topographic distribution and cytomorphologic features of stained cells varied considerably between classic and desmoplastic medulloblastomas. Four neoplastic phenotypes, representing gradations of neuronal differentiation, were identified: [Allegranza 1991] apolar, blast-like PCNA/cyclin(+) cells devoid of beta III reactivity (Nb1); [Bravo et al. 1987] apolar, often binucleated and/or fusiform, PCNA/cyclin (+) cells with pronounced beta III staining in their protoperikarya and their growth cones (Nb2); [Burger et al. 1987] beta III-immunoreactive immature polar neurons with varying degrees of neuritic development, reading to significant neuritogenesis in the "pale islands" of desmoplastic medulloblastomas (Nb3). The majority of Nb3 phenotypes were PCNA/cyclin (-), although subpopulations of such polar tumor cells exhibiting PCNA staining were also identified; and [Burger et al. 1991] beta III-immunoreactive, PCNA/cyclin (-) mature ganglion-like cells (Nb4). A high PCNA/cyclin labeling index (> 80%) was obtained in 20 poorly differentiated classic medulloblastomas while, significant intratumoral staining heterogeneity was observed in 23 cases of desmoplastic medulloblastomas and 3 cases of "medulloblastomas with ganglion cells": A high labeling index (LI)(> 80%) in the reticulin-impregnated poorly differentiated areas of tumor contrasted with sharp decline of PCNA staining and a very low LI (< 10%) in areas of overt neoplastic neuritogenesis ("pale islands") displaying strong beta III reactivity. Neoplastic ganglion cells were beta III (+)/PCNA (-). Our findings indicate that the majority of differentiating neuronal phenotypes undergoing cytomorphological changes of neuritic development (Nb3), and all neoplastic ganglion cells (Nb4 phenotypes) are PCNA (-), in contrast to actively proliferating, poorly differentiated, tumor cells that are PCNA (+). Although PCNA staining corresponded in part, to beta III (-) blast-like elements (Nb1), a co-expressive pattern of staining for beta III and PCNA/cyclin also was observed in subpopulations of poorly differentiated tumor cells (Nb2), indicating that transformed neuroblasts are capable of expressing differentiation-associated neuronal cytoskeletal proteins while still remaining in the proliferative compartment of the cell cycle. Our observations suggest that only neuritogenesis and acquisition of ganglionic phenotype are significant maturational events in medulloblastomas (indicating entry into the quiescent phase of the cell cycle) and provide further support for the neuronal lineage and differentiation potential of these cerebellar embryonal tumors.

Monoclonal anti-dipeptide antibodies cross-react with detyrosinated and glutamylated forms of tubulins.

Two monoclonal antibodies, GLU-1 and A1.6, raised against gamma-L-glutamyl-L-glutamic acid dipeptide (Glu-Glu) and Ca(2+)-dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu-Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross-reaction of the antibodies to the alpha-tubulin subunit. alpha-Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and A1.6 specifically recognize the detyrosinated form of alpha-tubulin. When microtubule protein purified from brain was probed, not only alpha-but also, to a lesser extent, beta-tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the gamma position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class III beta isotype at amino acid position 437 to 439, it is suggested that GLU-1 and A1.6 are able to recognize the glutamylated form of beta-tubulin. These results indicate that the C-terminal Glu-Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of alpha-tubulin and the glutamyl side chain of beta-tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post-translationally modified tubulin subunits.

Localization of the neuronal class III beta-tubulin isotype in foci of early neuritogenesis supports divergent neuroblastic differentiation potential in Wilms' tumors.

Wilms' tumors are embryonic neoplasms that have been proposed to originate from the metanephric blastema and are capable of divergent epithelial and mesenchymal differentiation. Neuroepithelial differentiation in these tumors remains controversial. The aim of this study was to examine the phenotypic profile of certain neuronal and glial antigenic determinants in a series of 21 Wilms' tumors. Immunohistochemical studies were performed by using monoclonal antibodies against the neuronal class III beta-tubulin isotype (beta III), the phosphorylated and phosphorylation-independent epitopes of neurofilament protein, and synaptophysin; antisera to gamma-enolase (neuron-specific enolase) glial fibrillary acidic protein, and S100 protein were also used. Foci of neoplastic cells with neurite-like processes that exhibited intense beta III staining were demonstrated in blastemalike areas of three of 21 tumors. In one case, Homer Wright rosettes (stained for beta III) were identified. Areas of abortive neuritic development were also labeled with antibodies to gamma-enolase. No reactivity was obtained in these foci for phosphorylated and phosphorylation-independent epitopes of neurofilament protein, synaptophysin, glial fibrillary acidic protein, and S100 protein. The remainder of the tumors (18 of 21) were negative with the panel of neural markers. Our results indicate that divergent neuroblastic differentiation, evidenced as early neoplastic neuritogenesis, may be present in the blastematous component of Wilms' tumor subsets.

Glomus cell differentiation in the carotid body region of chick embryos studied by neuron-specific class III beta-tubulin isotype and Leu-7 monoclonal antibodies.

Development of the carotid body and the glomus cell groups in the wall of the common carotid artery and its branches was examined in chickens at various developmental stages by immunohistochemistry using three different monoclonal antibodies, i.e., anti-neuron-specific class III beta-tubulin isotype (TuJ1), anti-rat brain beta-tubulin, and anti-Leu-7 (HNK-1) antibodies. All the antibodies reacted with neurons. The carotid body anlage was first discerned at 6 days of incubation at the lateral portion of the third branchial artery. The cells and nerve fibers immunoreactive for TuJ1, brain beta-tubulin and Leu-7, which were connected with the distal ganglion of the vagus nerve, were found around the carotid body anlage at this stage. Within the carotid body anlage, no immunoreactivity yet appeared. The immunoreactive cells were accumulated around the carotid body anlage until 8 days of incubation. From 9 days of incubation, the immunoreactive cells continuing with the distal vagal ganglion began to enter into the carotid body anlage and also dispersed widely along the common carotid artery and its branches, giving rise to the glomus cells. At 12 days of incubation, a large portion of the carotid body was occupied by the immunoreactive cells. Thus, the present study evidences that the glomus cells in the carotid body and around the arteries are emigrés that arrive in each residential place from the distal vagal ganglion. Immunoreactivity for TuJ1, brain beta-tubulin, and Leu-7 in the glomus cells started to decrease at late stages of embryonic development. After hatching, no TuJ1-immunoreactive cells were detected in the carotid body region.

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

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)

The stromal Schwann cell during maturation of peripheral neuroblastomas. Immunohistochemical observations with antibodies to the neuronal class III beta-tubulin isotype (beta III) and S-100 protein.

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III; analogous to the beta' 1-/beta 2-tubulin isoform) to the Schwann cell-associated S-100 protein focusing on topographic relationships of Schwann-like cells to differentiating neuronal phenotypes during stromal development in human peripheral neuroblastomas. The earliest appearance of Schwann cells in poorly differentiated (classical) neuroblastomas is heralded by S-100 protein-immunoreactive cells in close association with tumor blood vessels. In subsequent stages of maturation, i.e. maturing neuroblastoma (ganglioneuroblastoma and gangliocytoma), S-100 protein-positive cells are mostly confined to the connective tissue septa dividing tumor into lobules, and are not freely interspersed with beta III-immunoreactive neoplastic neurons. Significant ensheathment of individual axon-like processes by Schwann cells occurs only in mature ganglioneuromas. beta III is localized in a full spectrum of neoplastic neuronal phenotypes, ranging from poorly-differentiated apolar neuroblasts (often signaling ensuing neuritogenesis) to mature ganglion cells, but not in Schwann cells, or other cell types of the stroma. Our observations suggest that Schwann cells in peripheral neuroblastomas are stroma-derived cells and not an expression of divergent neoplastic differentiation.

Immunohistochemical localization of a neuron-specific beta-tubulin isotype in the developing chicken ultimobranchial glands.

The localization of a neuron-specific beta-tubulin isotype in the ultimobranchial glands from chickens at various stages of development was studied by means of light- and electron microscopic immunohistochemistry with a monoclonal antibody (TuJ1) against the beta-tubulin isotype, c beta 4. At 8 days of incubation, many C cells in the ultimobranchial glands showed immunoreactivity for TuJ1 invariable degrees, weak to intense. At 12 days of incubation, a vast majority of C cells were intensely immunoreactive for TuJ1, and further TuJ1-immunoreactive nerve fibers were distributed in the ultimobranchial glands. At 14 and 16 days of incubation, intense immunoreactivity for TuJ1 was sustained in the C cells. Electron microscopic analyses revealed that TuJ1 immunoreactivity was diffusely distributed throughout the cytoplasm and also localized on the secretory granules of C cells at these stages. TuJ1 immunoreactivity in the C cells started to decrease at late stages of embryonic development. At the hatching period, dense distributions of TuJ1-immunoreactive nerve fibers were observed in the ultimobranchial glands, whereas TuJ1 immunoreactivity of the C cells became very weak. In 10-day-old chickens, TuJ1 immunoreactivity was restricted to the nerve fibers.

Differential localization of class III, beta-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex.

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III) to that of calbindin-D28k in 40 human fetal and postnatal cerebella ranging from 12 weeks gestation to adulthood. In the external granule layer of the developing cerebellar cortex, beta III staining was present in the premigratory (postmitotic) zone of horizontal neurons but was absent in "epithelioid" cells of the subpial proliferative mitotic zone. In the molecular layer, intense beta III staining was associated with parallel fibers, stellate/basket neurons and migrating fusiform granule neurons. beta III staining was also present in internal granule neurons. In contrast, beta III was not detectable in fetal and neonatal Purkinje neurons and Golgi II neurons, but was evident in these neurons from juvenile and adult cerebella. Calbindin-D28k staining was present in Purkinje neurons also delineating their somatic spines ("pseudopodia"), lateralizing and apical dendrites (including dendritic spines), subpopulations of small to intermediate-sized Golgi II neurons in the internal granule layer ("synarmotic cells" of Landau), large to medium-sized subcortical Golgi II neurons and neurons of cerebellar roof nuclei, at various gestational stages and postnatally. It was absent in the external granule layer, parallel fibers, stellate/basket and internal granule neurons. Variable degrees of beta III and calbindin-D28k staining were detected in subpopulations of immature neuroepithelial cells of the ventricular matrix at the roof of the fourth ventricle. Glial (including Bergmann glia) and mesenchymal cells were not stained for either antigenic determinants. The differential expression of calbindin-D28k and beta III defines distinct populations of neurons in the developing human cerebellar cortex and supports the ontogenetic concept of Ramon y Cajal.

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 isotype, 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 31 days in vitro. Western blots on whole SDS extracts of cells 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, and 31 days) were probed with monoclonal antibodies (MoAb) specific for the above-mentioned microtubule proteins. For these and all other markers employed, immunoperoxidase histochemistry was performed only on the matrix cultures. The immunoblot experiments demonstrated that the Class 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 by immunohistochemistry. In contrast, explants from all culture conditions 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 organ culture system. Both sets of experiments indicate that these markers are not altered under three different conditions of growth over a one-month period in vitro. The expression of GFA protein and the absence of detectable levels of Class III beta-tubulin, MAP2, and tau are in keeping with the astrocytic phenotype of the C-6 cell line.

Even in culture, oligodendrocytes myelinate solely axons.

Cerebral hemispheres from mouse embryos at 15 days of gestation were dissociated and maintained in culture for several weeks in a medium which permitted homochronic and homotypic oligodendrocytes and neurons to interact in the presence of other central nervous system cells. After 13-14 days in culture a few oligodendrocytes changed from highly branched, "sun-like," nonmyelinating cells to sparcely branched myelinating cells. The number of fibers myelinated per oligodendrocyte ranged from 1 to 10, similar to that described previously in vivo in the corpus callosum. When an oligodendrocyte began to myelinate, it immediately myelinated a maximum number of fibers, suggesting that the number of axons to be myelinated by the oligodendrocyte was predetermined. When only one fiber was in the vicinity of a myelinating oligodendrocyte, whorls of myelin-like figures were seen at the tip of oligodendrocyte processes that had not reached an axon. Myelinated fibers were unambiguously identified as axons both by immunostaining and by electron microscopy. Myelin was not observed around astrocyte processes or around dendrites. The exclusive myelination of axons suggests the existence of a specific axonal recognition signal which attracts oligodendrocyte processes.