AMultiple-Loop , Double-CubeMicroarray Design Applied to Prostate Cancer Cell Lines withVariable Sensitivity to Histone Deacetylase Inhibitors

Purpose:Althoughmicroarray technologyhas beenwidely adoptedby 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 byWestern 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 z 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 HDACIinduced changes in gene expression possibly related to drug resistance. Histone deacetylase inhibitors (HDACI) are anticancer agents that inhibit the activity of enzymes that acetylate histone tails and other proteins. Acetylation is thought to play a role in cancer development and progression (1). Several HDACIs are in clinical development for solid malignancies. In preclinical studies, these agents have shown antitumor activity in vitro and in vivo in prostate cancer models. Phase I and II clinical trials with HDACI resulted in stable disease and partial responses in a wide range of solid malignancies, including prostate cancer (2, 3). Recently, the Food and Drug Administration approved HDACI vorinostat for the treatment of advanced cutaneous T-cell lymphoma and several clinical trials are ongoing in solid malignancies. Since its initial success as a tool for quantitative transcription level analysis in 1995 (4), microarray technology has become instrumental for global and parallel analysis of cellular biology (5). Although microarray technology has been rapidly adopted by the scientific community, experimental design and interpretation of the resulting data remain challenging. The choice of experimental design can be crucial in determining the extent to which biological and technical variation affect the results of a microarray experiment. Commonly used experimental designs in dual-color arrays include the ‘‘loop design’’ and ‘‘reference design. ’’ In the loop design, all samples are hybridized in such a way that the connected arrays form a loop pattern (1-2, 2-3, 3-4, and 4-1). In a reference design, all samples of interest are cohybridized with the same reference sample (1-R, 2-R, and 3-R; ref. 6). In this article, we report results of a microarray experiment conducted using a more complex design as a case study to increase understanding of the technical aspects in experimental design and analysis of high-throughput gene expression studies. Before our microarray experiment, we tested the sensitivity of prostate cancer cells to HDACIs. PC3 cells were more resistant than DU145 cells to HDACI treatment. To improve understanding of the mechanism underlying this difference in resistance, we designed an experiment to simultaneously compare global gene expression in 16 different biological samples with 22 arrays. To determine the relative contribution of three experimental Cancer Therapy: Preclinical Authors’Affiliations: Prostate Cancer Program and Divisionof ClinicalTrials and Biometry, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University; Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health; and Department of Pathology, Johns Hopkins School of Medicine, Baltimore, Maryland Received1/15/08; revised 6/5/08; accepted 6/18/08. Grant support: Prostate Cancer Foundation and National Cancer Institute Specialized Program of Research Excellence grant 58236 and Flight Attendant Medical Research Institute Grant. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints:Michael A. Carducci, ProstateCancer Program,The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-614-3977; Fax : 410-614-8160; E-mail: carducci@jhmi.edu. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-0119 www.aacrjournals. org Clin Cancer Res 2008;14(21) November1, 2008 6886 Research. on May 2, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals. org Downloaded from variables of interest on gene expression, all samples were hybridized according to a tailor-made ‘‘multidimensional loop’’ design. Independent comparison of all 16 samples was made possible with an adjusted ANOVA model for each feature on the array. This article discusses the pros and cons of our design and its feasibility for other researchers. Materials and Methods Cell culture and chemicals The following human prostate cancer cell lines (e.g., LNCaP, DU145, PC3, and CWR22R) were obtained from the American Type Culture Collection and cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum. Vorinostat [also known as suberoylanilide hydroxamic acid (SAHA)] was obtained from Aton Pharma/Merck and valproic acid (VPA) and trichostatin A (TSA) were obtained from Sigma-Aldrich. Proliferation assays DU145 and PC3 cells were plated in 96-well plates at 4,000 cells per well and CWR22R and LNCaP cells were plated at 10,000 cells per well. Per treatment arm, 8 wells were used. All cells were allowed to adhere for 24 h before treatment, except LNCaP, which was allowed to adhere for 48 h. At the start of the experiment, freshly prepared drug was added to each well and incubated for 48 h. The proliferative activity was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) kit (Promega; Cell Titer 96 Non-Radioactive Cell proliferation assay), which is based on reduction of tetrazolium salt MTT to colored formazan by mitochondrial enzymes present only in living, metabolically active cells (7). In congruence with the manufacturers’ description, cells were incubated for 4 h with the dye solution after treatment with HDACI followed by overnight incubation with the stop solution. To acquire a uniform color, a shaker was used. The absorbance at 570 nm was read and background was subtracted using the 650 nm reading and a blank reading. P values were calculated using the two-sample t test assuming equal variances (Student’s t test). All MTT assays were conducted in triplicate. Microarray experiment Experimental design. To increase our insight in the mechanism of action of HDACIs on prostate cancer cells and to gain insight in possible resistance mechanisms against these agents, we compared the transcription levels of PC3 and DU145 cell lines after treatment with vorinostat and VPA. For the microarray experiment, 1 Amol/L vorinostat and 1 mmol/L VPA were chosen as the treatment doses. It has been shown that these doses are achievable in humans. Forty-eight or 96 h treatment with either 1 Amol/L vorinostat or 1 mmol/L VPA in both DU145 and PC3 cell lines were compared. In total, we compared 16 conditions with 22 arrays. We used dualcolor microarrays to take advantage of the ability to hybridize two samples per array. We preferred direct comparison on the more important questions while also allowing answering additional questions via indirect comparison. Some samples were hybridized three or four times to facilitate the comparisons of interest. In a loop design, a larger number of edges between samples is associated with higher variance in the corresponding comparison (6). No assumptions were made concerning (in)dependence of our experimental conditions. Taking into account dye swaps and technical replicates, the microarray setup resulted in a double cube. The directed graph (8) in Fig. 2B gives a schematic overview of the experimental set up. Each arrow represents one microarray slide comparing expression between two RNAs and is considered a block in the experimental layout (6). The sample on the tail side of the arrow is labeled with Cy5 (red). The sample on the head side of the arrow is labeled with Cy3 (green). The variables of interest included cell line (DU145 and PC3), HDACI (vorinostat and VPA), and duration of treatment (48 and 96 h). Control samples within each cube use the solvent specific for the HDACI. All samples are labeled and hybridized at least once with Cy3 and once with Cy5 to correct for dye