Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.
Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.
Johnson
Paul H
PH
Department of Cancer Research, Berlex Biosciences, Richmond, California 94804-0099, USA. JohnsonPaulH@att.net
Walker
Roger P
RP
Jones
Steven W
SW
Stephens
Kathy
K
Meurer
Janet
J
Zajchowski
Deborah A
DA
Luke
May M
MM
Eeckman
Frank
F
Tan
Yuping
Y
Wong
Linda
L
Parry
Gordon
G
Morgan
Thomas K
TK
McCarrick
Meg A
MA
Monforte
Joseph
J
D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D021983:Cyclophilins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015195:Drug Design; D015972:Gene Expression Regulation, Neoplastic; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008297:Male; D008956:Models, Chemical; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013329:Structure-Activity Relationship; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D000097642:GADD45 Proteins
D000199:Actins; D022762:CCAAT-Enhancer-Binding Proteins; D004396:Coloring Agents; D021983:Cyclophilins; D004247:DNA; D018840:HSP70 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011506:Proteins; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase; D013778:Tetrazolium Salts; D013844:Thiazoles; D051742:Transcription Factor CHOP; D014157:Transcription Factors; D000097642:GADD45 Proteins; C497249:DDIT3 protein, human; C022616:thiazolyl blue