Abstract
BackgroundPrimary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines.MethodsCultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis.ResultsWe show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis.ConclusionsThe recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first evidence that altered primary cilium expression and function may be part of some malignant phenotypes. Further, we provide the first evidence that ciliogenesis is not an all or none process; rather defects can arrest this process at various points, particularly at the stage subsequent to basal body association with the plasma membrane.
Highlights
Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression
Human astrocytes express primary cilia that reside within a membrane bound channel termed the "cilium-pit" that is the site of endocytosis Antibodies to acetylated tubulin, glu tubulin and adenylyl cyclase III (ACIII) are markers of primary cilia [34,35,36,37,38] and detected primary cilia in cultured log-phase human astrocytes (Figure 1)
Stage 1 was characterized by the presence of two vesicles at the distal end of the basal body; Electron Microscopy (EM) Cells were seeded into 35 mm dishes grown to confluence as monolayers over two days, washed in phosphate buffered saline (PBS) and fixed in 3% glutaraldehyde in Millonig's phosphate buffer for 1 hour at room temperature (RT)
Summary
Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. The expression and function of primary cilia in cancer cells has become a focus of attention but has not been studied in astrocytomas/glioblastomas. Certain proteins involved in cell cycle progression are linked to primary cilium expression (for review see [18,19,20]). Primary cilium region in cultured astrocyte cells is structurally complex and includes foci for endocytosis-based signaling. This indicates that there is a spatial link between receptor pathways associated with endocytosis and the primary cilium microenvironment. We noted several defects affecting the structure of astrocytoma/glioblastoma centrioles that were not observed in primary human astrocytes. We conclude that aberrant ciliogenesis is a common defect found in cells derived from astrocytomas/glioblastomas and this deficiency likely contributes to the phenotype of these malignant cells
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