Multiplex analysis of anti-apoptotic BCL2 family and caspase 3 activation by microbead arrays

Assay Drug Dev Technol. 2014 Apr;12(3):190-6. doi: 10.1089/adt.2013.557.

Abstract

We have developed a multiplex assay to measure the expression of anti-apoptotic proteins and caspase 3 activation using the Luminex platform. In this report, we show three applications for this assay. First, we used this assay to identify biomarkers for BCL2 inhibitors to obtain a quantitative measure of expression of anti-apoptotic proteins (BCL2, BCLxL, and MCL1) in a panel of cell lines and correlated their response to BCL2/BCLxL inhibitor, ABT-263 (navitoclax). Second, we used this assay to monitor the change of MCL1 protein expression and induction of active caspase 3 after treatment with cyclin-dependent kinase inhibitor flavopiridol. Finally, we used this assay to screen for small molecules that decrease MCL1 protein and identified new combinations with ABT-263. This method provides a quick and convenient way to measure basal expression of the anti-apoptotic proteins and monitor expression change upon drug treatment. It is also applicable for high-throughput screening for compounds that decrease the expression of these anti-apoptotic proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Carcinoma, Non-Small-Cell Lung / metabolism*
  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Caspase 3 / metabolism*
  • Cell Line, Tumor
  • Enzyme Activation
  • Gene Expression Profiling / methods*
  • Humans
  • Luminescent Measurements / methods*
  • Microspheres
  • Protein Interaction Mapping / methods*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*

Substances

  • Proto-Oncogene Proteins c-bcl-2
  • Caspase 3