Back to Archived Journals » International Journal of High Throughput Screening » Volume 1

Development and validation of an RNAi screen for ABT-737 sensitizers

Authors Yip KW, Kim I, Garrison J, Cortez A, Cheung N, Aza-Blanc P, Konopleva M, Andreeff M, Reed J

Published 20 September 2010 Volume 2010:1 Pages 163—170


Review by Single anonymous peer review

Peer reviewer comments 5

Kenneth W Yip1, Inki Kim1, Jason Garrison1, Apple Cortez1, Nancy Cheung1, Pedro Aza-Blanc1, Marina Konopleva2, Michael Andreeff3, John C Reed1
1Sanford-Burnham Medical Research Institute, La Jolla, CA, USA; 2Department of Leukemia, 3Department of Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX, USA

Abstract: The chemical compound ABT-737 is a nanomolar inhibitor of several antiapoptotic Bcl-2 family members with potential therapeutic efficacy for a variety of cancers. Herein, we describe the development of a complementation-based RNAi assay that can be used to identify mechanisms of ABT-737-resistance. HeLa cells, which were resistant to ABT-737, were optimized for reverse-transfection efficiency and tested for siRNA-mediated silencing. The developed assay utilized HeLa cell reverse-transfection with 10 nM siRNA, followed by 48 h incubation, ABT-737 or DMSO treatment for 24 h, and cell viability measurement using ATPlite (which measures ATP levels as an indicator of cell viability). As a validation, the kinase subset of the Ambion Silencer Human Druggable Genome siRNA Library V2, which consisted of 865 genes (three siRNA sequences per gene), was screened. Several assay-positive siRNAs were tested and confirmed to sensitize cells to ABT-737. Transfection of cells with siRNAs targeting Bcl-2 family member Mcl-1 also potently sensitized HeLa cells to ABT-737. The current assay thus represents a screen that can be utilized to identify ABT-737-sensitizing siRNAs and correspondingly, potential new targets for drug discovery.

Keywords: ABT-737, Bcl-2, apoptosis, RNAi screen, siRNA

Creative Commons License © 2010 The Author(s). This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.