BETi/HDACi treatment of CTCL cells induces significant alterations of transcripts regulating CTCL apoptosis
Based on our prior efficacy and toxicity studies, we selected 125nM birabresib (OTX015) (BETi) and 1nM romidepsin (HDACi) for all experiments in this report (Zhao et al., 2019). Gene expression was analyzed in triplicate on HH, Hut78, MyLa, SeAx and SZ4 CTCL lines at 0, 6, 48 and 96 hours post-treatment and normalized to housekeeping genes. Analysis was enriched for pro- and anti-apoptotic, oncogenic and tumor suppressor genes. Reference Zhao L, Ohkovat J-P, Hong EK, Kim YH, Wood GS. Preclinical studies support combined inhibition of BET family proteins and histone deacetylases as epigenetic therapy for cutaneous T-cell lymphoma. Neoplasia 21: 82-92, 2019.
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RT2 Profiler™ PCR Arrays (PAHS-012Z and PAHS-502Z, QIAGEN, Valencia, CA) were used to screen for changes in expression of 157 unique human genes in response to BETi/HDACi treatment. This gene set was enriched for apoptosis genes, oncogenes and tumor suppressor genes. All five CTCL lines were analyzed individually at 0, 6, 48 and 96 hours post-treatment. RNA was extracted from CTCL cells using RNeasy Kits (QIAGEN, Valencia, CA) following the manufacturer's instructions. Purified messenger RNA (mRNA) (2 μg) was used for the first-strand complementary DNA (cDNA) synthesis using iScript cDNA synthesis kit (Bio-Rad Labs, Hercules, CA). Quantitative RT-PCR was performed using the CFX96 Real-Time PCR System following the manufacturer's instructions. Each array contained five separate housekeeping genes (ACTB, B2M, GAPDH, HPRT1, and RPLP0) that were used for normalization of the data. Microarray data was normalized against the house keeping genes by calculating the ΔCt for each gene. Heat maps with row Z-scores of gene expression post-treatment over time, tabulation of fold regulation of gene expression at 96 hours post-treatment and volcano plots of gene expression at 96 hours post-treatment were generated and analyzed by using QIAGEN RT2 Profiler PCR Array Data Analysis v3.5. Each of 5 cell lines had triplicate samples for all transcripts. Fold-change (2^(-ΔΔCT)) is the normalized gene expression (2^(- ΔCT)) in the test sample divided the normalized gene expression (2^(- ΔCT)) in the control sample. For fold-changes >1, fold-change and fold-regulation values are equivalent. For fold-changes < 1, fold-regulation is the negative inverse of the fold-change. Fold-regulation alterations with p<0.05 were grouped into four color-coded ranges: ≥ 2x decrease (blue), 1.5-1.9X decrease (green), 1.5-1.9X increase (orange) and ≥ 2X increase (red). The p value calculations were based on a Student’s t-test of the replicate 2^(- ΔCT) values for each gene in the control group and treatment groups The p-value calculation used is based on parametric, unpaired, two-sample equal variance, two-tailed distribution.