CCRF-CEM Prednisolone treatment at 4h and 72h

It has been shown previously that glucocorticoids exert a dual mechanism of action, entailing cytotoxic, mitogenic as well as cell proliferative and anti-apoptotic responses, in a dose-dependent manner on CCRF-CEM cells at 72 h. Early gene expression response implies a dose-dependent dual mechanism of action of prednisolone too, something reflected on cell state upon 72 h of treatment. In this work, a generic, computational microarray data analysis framework is proposed, in order to examine the hypothesis whether CCRF-CEM cells exhibit an intrinsic or acquired mechanism of resistance and to investigate the molecular imprint of this, upon prednisolone treatment. The experimental design enables the examination of both the dose (0 nM, 10 nM, 22 uΜ, 700 uΜ) effect of glucocorticoid exposure and the dynamics (early and late, namely 4 h, 72 h) of the molecular response of the cells at the transcriptomic layer. In this work we demonstrated that CCRF-CEM cells may attain a mixed mechanism of response to glucocorticoids, however, there is clear evidence predicating towards an intrinsic mechanism of resistance. More specifically, at 4 h prednisolone appeared not to perform its expected function by down-regulating apoptotic genes, which is re-enforced by mechanisms, which down-regulate other sets of apoptotic genes. Also, low and high prednisolone concentrations up-regulates metabolic and signal-transduction related genes in both time points, thus grounding for a cell proliferation machinery. In addition, regulation of NF-κB-related genes implies an inherent mechanism of resistance through the established link of NF-κB inflammatory role and GC-induced resistance. The analysis framework applied here allows derivation of regulatory mechanisms activated by prednisolone through identification of early responding sets of genes. On the other hand, study of the prolonged exposure to glucocorticoids (72 h exposure) highlights the effect of homeostatic feedback mechanisms of the treated cells. Overall, it appears that CCRF-CEM cells in this study exhibit a diversified, combined pattern of intrinsic and acquired resistance to prednisolone, yet with a tendency towards inherent resistant characteristics, through activation of different molecular courses of action. For the assay of mRNA levels two sets of microarray chips were used (1,200 and 4,800 genes), obtained from TAKARA (Human Cancer Chip v.40 and IntelliGene™ II Human CHIP 1 respectively) (Chung, Park et al. 2004). The Human Cancer Chip v.40 platform is available in the GEO database under the accession number GPL13250 and the IntelliGene™ II Human CHIP 1 is available with the accession number GPL13251. Hybridization was performed with the CyScribe Post-Labeling kit (RPN5660, GE Healthcare, former Amersham Inc.) as described by the manufacturer. The fluorescent dyes used were Cy3 and Cy5. cDNAs were purified with PCR product clean-up kit (# 28104, QIAGEN Inc.). Slides were activated at 55o C for 30 min in 1% BSA. Samples were applied on the slides, and let to hybridize overnight at 55o C. The following day, slides were washed in 200 ml 0.1× SSC and 0,1% SDS for 3× 5 min, in 200 ml 0.1× SSC for 2× 5 min and in 200 ml ddH2O for 30 sec. Slides were dried by centrifugation at 1500 rpm for 3 min and scanned with a microarray scanner. Slides were scanned with a microarray scanner (ScanArray 4000XL) (Perkin Elmer Inc. MA, former GSI Lumonics, USA). Images were generated with ScanArray microarray acquisition software (GSI Lumonics, USA). Image analysis and raw data acquisition were performed with the ImaGene® software from BioDiscovery (California, USA). Microarray experimental design consisted of a small loop-design at 4h as described previously (Churchill 2002; Altman and Hua 2006). Our design and experiments were described previously (Lambrou, Vlahopoulos et al. 2009). Briefly, there were three sets of experiments at 4h designated as follows: 0vs1: control vs. 10nM prednisolone, 1vs3: 10nM prednisolone vs. 700uM prednisolone and 0vs3: control vs. 700uM prednisolone. This loop-design had the following property. The sum of the ratios of the 0vs1 and 1vs3 experiments should theoretically equal the ratio in the 0vs3 experiment. In the following sections we describe how this was used in order to filter genes. Experiments at 72h were based on a simple reference design. In particular, the experimental setups consisted of the five following pairs: control vs. 10 nM prednisolone at 4 h (designated as ‘1’), 10 nM vs. 700 uM prednisolone at 4 h (designated as ‘2’), control vs. 700 uM prednisolone at 4 h (designated as ‘3’), 22 uM vs. 700 uM prednisolone at 72 h (designated as ‘4’), and control vs. 700 uM prednisolone at 72 h (designated as ‘5’). The 4h experiments were performed in triplicates and the 72h experiments in single experiments. Creation of a Common Gene Set: Due to the derivation of different genes in each of the five experimental setups and the different number of array features between the two microarray chips, the clustering included genes present in all setups, forming a common gene set. This resulted in strict filtering of huge numbers of genes, since only few are present simultaneously in all five experiments. In total 586 genes were common in both platforms and these were used for further analyses. Identification of Differentially Expressed Genes: In order to identify potentially differentially expressed (DE) genes between samples and among genes of the same experiment we have used a one-sample z-test and two-sample z-test (Yang, Chen et al. 2002). The DE genes per experiment were identified at a confidence level of 95%. The False Discovery Rate (FDR) has been calculated as described previously (Klipper-Aurbach, Wasserman et al. 1995; Storey and Tibshirani 2003; Storey and Tibshirani 2003).