A Multiple-Loop, Double-Cube Microarray Design Applied to Prostate Cancer Cell Lines with Variable Sensitivity to Histone Deacetylase Inhibitors

Purpose: Although microarray technology has been widely adopted by the scientific community, analysis of the ensuing data remains challenging. In this article, we present our experience with a complex design microarray experiment on resistance mechanisms of histone deacetylase inhibitors (HDACI). Experimental Design: To improve our understanding of the underlying mechanism of HDACI resistance in prostate cancer cells, we designed a novel “multiple-loop, double-cube” cDNA microarray experiment. In the experiment of 22 arrays, DU145 and PC3 cells were treated with two different HDACIs (vorinostat and valproic acid) and incubation periods (48 and 96 h). Preprocessing included exploratory analyses of the quality of the arrays and intensity-dependent within-array Loess normalization. An ANOVA model was used for inference. The results were validated by Western blot analysis of known treatment targets. Results: Treatment of PC3 and DU145 cells with HDACIs caused 2.8% to 10% (P < 0.001) differential expression across conditions; 51% to 73% of these genes were up-regulated and 28% to 49% were down-regulated. The extent of differential expression was associated with cell line (DU145 > PC3), HDACI (valproic acid ≥ vorinostat), and duration of treatment (96 > 48 h). We identified known and new treatment targets involved in cell cycle and apoptosis. Conclusion: A multiple-loop, double-cube microarray design can be used to identify HDACI-induced changes in gene expression possibly related to drug resistance.

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