Transcriptional Profiling of the Dose Response: A More Powerful Approach for Characterizing Drug Activities

Abstract

The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901) across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression

“…Experimental design, cell growth, and treatment for expression profiling analysis were as previously described (19). Four lung cancer cell lines (DMS53, H2227, H1048, and H1694) were plated in 96-well plates, and JQ1 in 3-fold serial dilution covering six logarithm ranges in drug concentration was added 24 hours later.…”

“…The microarray data have been deposited into the ArrayExpress database under the accession number E-MTAB-3449 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3449). The Sigmoidal Dose Response Search (SDRS) algorithm was applied as previously described (19), with the modification that a 1.1 multiple and no step function was used to set the 128 logevenly distributed test values for C. For data visualization, the 12 intensity values for each treatment were scaled from 0 to 1, providing a view of the direction and consistency of change for each probe set. Note that where a consistent dose response is not present, this scaling result in a random pattern of high and low values across the dose series.…”

“…The treatment time was limited to 2 hours to minimize detection of expression changes that are secondary or indirect. The SDRS algorithm (19) was applied to the microarray data, and it identified probe sets that show dose-dependent regulation by JQ1. The probe sets were filtered for those that showed !2-fold change with EC 50 < 750 nmol/L in at least one cell line, with a statistical cutoff of P < 0.05.…”

A Negative-Feedback Loop between the Detoxification/Antioxidant Response Factor SKN-1 and Its Repressor WDR-23 Matches Organism Needs with Environmental Conditions

“…For example, does feedback predominantly impact maximal activity levels or affinity for a signal or both? Relationships between signal strength and feedback could be complex given that the activity of the transcription factor and thus activation of the repressor gene are both expected to increase nonlinearly with increasing signal (52). It is also unclear if feedback exists in the absence of an external signal, when the activity of many stress-inducible transcription factors is low.…”

Toxicogenomics for transcription factor-governed molecular pathways: moving on to roles beyond classification and prediction

“…This approach provided an aggregate BMD value for significantly enriched genes within various GO categories, including biological processes or molecular functions. There are other dose-response analysis tools available to estimate BMD or an effective concentration causing 50 % response-EC50 (Burgoon et al 2009;Ji et al 2009). Many genomic studies have made use of these tools for assessing doseresponse (e.g., Black et al 2012;Kopec et al 2010;Thomas et al 2011).…”