Altered Cellular Distribution and Subcellular Sorting of γ-Tubulin in Diffuse Astrocytic Gliomas and Human Glioblastoma Cell Lines

Abstract

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.

Role of the Centrosomal MARK4 Protein in Gliomagenesis

“…CIN is a general property of aneuploid cancer cells and is generated by defects in different processes, among which the regulation of the number of centrosomes, the dynamics of microtubules attachment to the kinetochores and the overall control of cell cycle. Defects in centrosomal number and structure have been well documented in gliomas Katsetos et al, 2006;Magnani et al, 2009) raising the issue whether the increased MARK4L isoform, a gene involved in microtubule dynamics, may concur to errors in chromosomal segregation driving gliomagenesis. Recent application of multidimensional technological approaches has comprehensively highlighted the scenario of glioma genes and core pathways.…”

“…Multipolarity in cancer cells is considered an essential transient stage prior to clustering extra centrosomes in a bipolar fashion (Brinkley, 2001). Multiple centrosomes have been detected in many types of cancer cells including glioma ( Figure 4) and strongly linked to aneuploidy in a variety of studies Ganem et al, 2009;Ghadimi et al, 2000;Katsetos et al, 2006;Lingle et al, 2002;Magnani et al, 2009;Pihan et al, 1998). A positive linear correlation between the p e r c e n t a g e o f c e l l s w i t h s u p e r n u m e r a r y centrosomes and the extent of aneuploidy within a panel of glioblastoma cell lines is shown in Figure 5.…”

Centrosome abnormalities in giant cell tumour of bone: possible association with chromosomal instability

“…In addition to the classic punctate arrangement, there are several types of other aberrant centrosome patterns that have been reported in neoplastic cells, such as centrosome enlargement, string-like, V-shaped and sand-like centrosome, as well as diffuse pattern. 14,8 In this study, only the punctate pattern was taken into account, as this was considered to be the optimal way of detecting centrosome aberrations on formalin-fixed paraffin-embedded or frozen tissue. Cells containing one or two centrosomes were considered negative, whereas those containing more than two centrosomes were regarded as positive (that is, showing centrosome amplification).…”

“…It has a key role in cytoplasmic division and regulation of cell-cycle progression, as well as in the establishment of an interphase cytoplasmic microtubule network and bipolar mitotic spindles. 8 Normal diploid somatic cells contain a single centrosome that must duplicate once before the next mitosis. Thus, at any given time point during the cell cycle, cells have either one unduplicated or two duplicated centrosomes.…”

Genetic Diversity of Glioblastoma Multiforme: Impact on Future Therapies

“…Several studies point to a link between centrosome amplification, chromosomal instability and the development of cancer (for review see: D´Assoro et al, 2002). Cells in resected high grade gliomas and cultured glioblastoma cells have been reported to exhibit often centrosome amplifications (Loh et al, 2010) and the centrosomal protein -tubulin is overexpressed and shows altered subcellular localization in GBM (Katsetos et al, 2006;Loh et al, 2010). Multipolar mitoses were occasionally observed in time lapse recordings of cultured glioblastoma cells (Hegedüs et al, 2000).…”