ecdysterone and Glioma

Transcription control systems enable experimental regulation of transgene expression in eukaryotic cells by application of specific repressors or inducers. Currently used inducible systems include tetracycline (tet-off), dimerizer and ecdysone systems. While numerous studies have utilized a single system, their comparative performance under identical conditions remains unclear. We therefore compared the efficiency of these three systems in C6 glioma cells by using transient transfection and the lacZ reporter gene. Highest induced activity was found with the ecdysone system, followed by tet-off and dimerizer systems. Both lowest repressed activity and highest regulation were revealed with the dimerizer system, followed by ecdysone and tet-off systems. Our data suggest that the most appropriate system depends on the experimental procedures, the application and the gene to be regulated.

Topics: Animals; Dimerization; Ecdysone; Ecdysterone; Gene Expression Regulation, Neoplastic; Glioma; Protein Synthesis Inhibitors; Rats; Tetracycline; Transfection; Tumor Cells, Cultured

The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.

Topics: Bradykinin; Brain Neoplasms; Cell Adhesion; Culture Media, Serum-Free; Cyclin-Dependent Kinase Inhibitor p16; Ecdysone; Ecdysterone; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, p16; Glioma; GTP-Binding Proteins; Humans; Insulin; Integrins; Neoplasm Proteins; Promoter Regions, Genetic; Pseudopodia; Recombinant Fusion Proteins; Signal Transduction; Tumor Cells, Cultured