Identification of Prostate Cancer-Related Genes Using Inhibition of NMD in Prostate Cancer Cell Lines
Final rept. 1 Jan 2004-31 Dec 2006
HEALTH RESEARCH INC BUFFALO NY
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A strategy to identify mutant genes using inhibition of nonsense mediated decay NMD in cell lines has been proposed by others. Blocking translation with antibiotic emetine has been shown to inhibit the NMD. Stabilization of mutant mRNA following the inhibition of NMD with emetine can be detected using microarray technology, such as Affymetrix genechips, for example. Unfortunately, too many genes that do not contain any mutations show mRNA increase following emetine treatment due to stress response to the inhibition of translation or due to being a natural substrate for NMD, thus complicating the identification of mutant genes. We have developed a modification of the method which includes inhibition of NMD using two alternative methods. First is to inhibit translation with emetine. Second is to block NMD by inhibiting SMG-1 kinase with caffeine and than to block transcription with actinomycin D and either to continue blocking NMD with caffeine or to activate NMD by caffeine withdrawal. Analyzing the mRNA accumulation following emetine treatment as well as mRNA degradation following blocking of transcription in the presence or absence of NMD with the Affymetrix analysis using simple analytical algorithm allows selection of candidate genes for sequencing with high efficiency. Using our method of NMD inhibition we have identified several genes containing bi-allelic inactivating mutation in prostate cancer cell lines.
- Genetic Engineering and Molecular Biology
- Anatomy and Physiology
- Medicine and Medical Research