RETINOIC ACID INDUCED DISRUPTION OF cAMP-INDUCED HUMAN SVG ASTROGLIAL CELL DIFFERENTIATION: MORHOLOGY AND GLOBAL GENE EXPRESSION EFFECTS.

L D Burgoon1,3, K Y Kwan2,M R Fielden2,3, J E Trosko3,4, T R Zacharewski2,3. 1Dept. of Pharmacology & Toxicology; 2Dept. of Biochemistry & Molecular Biology; 3Institute for Environmental Toxicology; National Food Safety & Toxicology Center; 4Dept. of Pediatrics & Human Development, Michigan State University, East Lansing, MI, USA.

Previous work has shown that treatment of human SVG cells with 5 µM forskolin (F) and 200 µM 3-isobutyl-1-methylxanthine (iBMX) increases cAMP levels resulting in differentiation and dramatic morphologic changes. We show that co-treatment with 0.5 µM retinoic acid (RA) enhances these morphologic changes as measured by increases in the total number of processes (p = 0.03) and the length of processes contacting an adjacent cell (LPT; p < 0.01) over time. At 36h, RA inhibited regression of LPT (p = 0.02) with increases in mean process length (p = 0.04) and the longest primary process (p <0.01). A custom, SVG-specific cDNA microarray (2,990 genes) was used to investigate the temporal changes in gene expression during cAMP induced differentiation. Temporal changes were identified using two different techniques. First, an incomplete block design, individual gene mixed-model ANOVA was used for analysis, followed by t-tests using a step-down Bonferroni adjustment for multiple comparisons. The mixed model included treatment, time, dye and treatment x time as fixed effects, and spots as random effects. To identify genes with significant expression changes during differentiation, least squares means of F/iBMX treated cells were compared to time-matched vehicle control samples utilizing t-tests. For comparison, a second technique utilizing Shannon Entropy filtering of normalized microarray data from treated, vehicle control, and untreated/time 0 h control cells, with Principal Components Analysis (PCA) of the filtered data to result in the identification of expression patterns was used. Results from the two divergent approaches were comparable and identified significant temporal and treatment effects on gene expression, although some differences were observed.