Expression of Nedd5, a mammalian septin, in human brain tumors

Abstract

The septins are a family of cytoskeletal GTPases that play an essential role in cytokinesis in yeast and mammalian cells. Nedd5 is a mammalian septin known to associate with actin-based structures such as the contractile ring and stress fibers. In the present study, we examined the expression of Nedd5 in a series of human brain tumor cell lines and surgical specimens by northern and western analyses. The temporal expression of Nedd5 in U373 astrocytoma cells at various timepoints throughout the cell cycle was determined by an analysis of lovastatin- and nocodazole-treated, synchronized cell populations. The intracellular localization of Nedd5 was determined by immunocytochemistry of steady state cultures and nocodazole-treated cultures enriched in M phase cells. The effects of inhibiting Nedd5 expression in human brain tumors was determined by stably transfecting U373 astrocytoma cells with an antisense-Nedd5 cDNA expression vector and by analyzing clones for Nedd5 expression by immunocytochemistry, morphological changes, cell growth and nuclear content. All human brain tumor cell lines and surgical specimens expressed Nedd5 transcript and protein. Synchronized U373 astrocytoma cells showed a relative increase in Nedd5 transcript levels from late Gl to G2M phases; and an increase in Nedd5 protein levels from S to G2M phases. Maximum expression of both transcript and protein levels was observed at the G2M phase. By immunocytochemistry, Nedd5 was concentrated at the cleavage furrow of mitotic cells. Double staining with Nedd5 and F-actin showed co-localization of Nedd5 with actin filaments except during cytokinesis. Antisense-Nedd5 expression led to an accumulation of nuclear content. These data suggest that Nedd5 is involved in the process of cytokinesis in human brain tumours. Nedd5 expression may be cell cycle-dependent with increased levels found at G2M phase. Blocking Nedd5 expression in astrocytoma cells by antisense interferes with the process of cytokinesis during cell division.