It is known that application of TSPO ligands as well as knockdown of the mitochondrial 18 kDa translocator protein (TSPO) modulate viability, proliferation, adhesion, and migration of glioblastoma cells, as well as angiogenesis.
More...It is known that application of TSPO ligands as well as knockdown of the mitochondrial 18 kDa translocator protein (TSPO) modulate viability, proliferation, adhesion, and migration of glioblastoma cells, as well as angiogenesis. To study the ability of the TSPO to regulate gene expression in relation to these functions we applied microarray analysis of gene expression to U118MG glioblastoma cells. Seen at the time points of 15, 30, and 45 minutes, the TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. These changes also included gene products that are part of the canonical pathway for modulation of general gene expression. These changes peaked at 30 minutes. Thus, it appears that the TSPO is part of the retrograde mitochondrial-nuclear signaling pathway for modulation of gene expression. Consequently, our data indicate that this is a major venue whereby TSPO may drive its numerous functional effects. Keywords: modulation of nuclear gene expression, mitochondrial 18 kDa translocator protein (TSPO), TSPO ligand, PK 11195, 2-Cl-MGV-1, retrograde mitochondrial-nuclear signaling pathway, microscopy, mitochondria, cell nucleus
abstract of annual meetingof the israel society for neuroscience, section B, abstract # 98.
Overall design: The aim of the study was to compare the gene expression profile using human expression beadchip of 4 experimental groups in triplicates: Vehicle (Base line (BL) - Without any treatment), 15 min after stimulus, 30 min after stimulus and 45 min after stimulus. The experiment design was to compare the change in expression during a time course of 0,15,30,& 45 min with 3 replicates for each time point. First of all, U118MG cells (1.3 × 10^6) were seeded in Petri dishes (i.e. 28.5 × 10^3 cells / cm2) and allowed to proliferate for 3 days in full medium. Experiments for gene expression assayed with microarray typically consisted of three experimental groups and a vehicle control group (n = 3 for each group). After seeding and proliferation, serum deprived medium was applied for 24 hrs, in the experimental groups ending with the inclusion a choice of various PK 11195 exposures i.e. either 15 min, 30 min, or 45 min. Exposure to PK 11195 implies serum deprived medium with 1% alcohol (vehicle) and PK 11195 (25 µM final concentration) for the required time period. 25 µM of PK 11195 is the optimal concentration for these types of experiments with U118MG cells (Kugler et al., 2008). The cells were collected by trypsinization, washed by centrifugation in phosphate buffered saline (PBS) (400 × g, 5 min), and lysed [RLT lysis buffer provided with the RNeasy Mini Kit diluted with β-mercaptoethanol (1 : 100)], according to the manufacturer's instructions. Lysates were stored at -70oC.
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