Biological Systems Engineering

 

Date of this Version

2011

Citation

Biotechnol Bioeng. 2011 February ; 108(2): 395–403.

Comments

© 2010 Wiley Periodicals, Inc. Used by permission.

Abstract

Identifying molecular mechanisms or therapeutic targets is typically based on large-scale cellular analysis that measures the abundance of mRNA or protein; however, abundance does not necessarily correlate with activity. We report a method for direct large-scale quantification of active pathways that employs a cellular array with parallel gene delivery of constructs that report pathway activity. The reporter constructs encode luciferase, whose expression is influenced by binding of transcription factors (TFs), which are the downstream targets of signaling pathways. Luciferase levels are quantified by bioluminescence imaging (BLI), which allows for rapid, noninvasive measurements. Activity profiles by BLI of 32 TFs were robust, consistent, and reproducible, and correlated with standard cell lysis techniques. The array identified five TFs with differential activity during phorbol-12-myristate-13-acetate (PMA)-induced differentiation of breast cancer cells. A system for rapid, large-scale, BLI quantification of pathway activity provides an enabling technology for mechanistic studies of cellular responses and processes.