Cellular arrays for large-scale analysis of transcription factor activity.
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, non-invasive 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.
Bellis
Abigail D
AD
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208-3120, USA.
Peňalver-Bernabé
Beatriz
B
Weiss
Michael S
MS
Yarrington
Michael E
ME
Barbolina
Maria V
MV
Pannier
Angela K
AK
Jeruss
Jacqueline S
JS
Broadbelt
Linda J
LJ
Shea
Lonnie D
LD
D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D046228:Microarray Analysis; D014157:Transcription Factors / Q000378:metabolism*
D014157:Transcription Factors; D008156:Luciferases
Biotechnol Bioeng. 2007 Oct 1;98(2):486-97
17486653
Annu Rev Cell Biol. 1994;10:405-55
7888182
Cell Growth Differ. 1999 Oct;10(10):677-83
10547071
Bioinformatics. 2006 Oct 15;22(20):2516-22
16908499
Lab Chip. 2007 Jan;7(1):77-85
17180208
Cancer Chemother Pharmacol. 2009 Mar;63(4):673-80
18597088
Biotechnol Bioeng. 2004 Jun 30;86(7):827-34
15162459
Genes Dev. 2001 Sep 15;15(18):2343-60
11562345
Cancer Res. 1998 Jun 1;58(11):2335-8
9622069
Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10907-12
18669654
Cancer Res. 1987 Mar 15;47(6):1615-20
3102047
Mol Cell Endocrinol. 1992 Dec;90(1):77-86
1301400
Cell. 2006 Aug 25;126(4):663-76
16904174
N Engl J Med. 1996 Jan 4;334(1):28-33
7494569
Nat Protoc. 2007;2(2):392-9
17406600
Brain Res Mol Brain Res. 1998 Jan;53(1-2):130-7
9473629
J Exp Med. 1998 Jun 15;187(12):2031-6
9625762
Science. 1997 Sep 5;277(5331):1508-10
9278514
Behav Brain Res. 2001 Nov 1;125(1-2):279-84
11682119
Toxicology. 2002 Dec 27;181-182:131-41
12505298
Lab Invest. 2006 Sep;86(9):867-72
16865089
Mol Cancer Res. 2007 Jan;5(1):71-85
17259347
Nat Rev Genet. 2006 Mar;7(3):200-10
16485019
Genet Epidemiol. 2002 Feb;22(2):170-85
11788962
Trends Endocrinol Metab. 2002 Mar;13(2):55-60
11854019
EMBO J. 1994 Nov 15;13(22):5421-32
7957108
Cell. 2002 Feb 22;108(4):439-51
11909516
Stat Appl Genet Mol Biol. 2004;3:Article3
16646809
Bioinformatics. 2003 Jan 22;19(2):185-93
12538238
J Cell Physiol. 1990 Oct;145(1):162-72
2211839
Protein Eng. 1999 Dec;12(12):1035-40
10611396
Nat Methods. 2008 Mar;5(3):253-60
18297081
Oncogene. 2007 Apr 2;26(15):2212-9
17401430
Oncogene. 2005 Jun 16;24(26):4193-205
15806162