Abstract
BackgroundGene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. An increase in transcription in response to NMD inhibition of a subset of genes is a major cause of false positives when genes are selected for sequencing analysis. To distinguish between mRNA accumulations caused by stress response-induced transcription and nonsense-containing mRNA stabilizations is a challenge in identifying mutant genes using GINI.MethodsTo identify potential tumor-suppressor genes mutated in prostate cancer cell lines, we applied a version of GINI that involves inhibition of NMD in two steps. In the first step, NMD is inhibited in duplicate tissue-culture plates. During this step, both the substrate for NMD and stress-response mRNA transcripts are accumulated in cells. In the second step, transcription is inhibited in both plates and NMD is inhibited in one plate and released in the second plate. Microarray analysis of gene-expression profiles in both plates after the second step detects only the differences in mRNA degradation but not in mRNA accumulation.ResultsAnalyzing gene expression profile alterations in 22RV1 and LNCaP prostate cancer cells following NMD inhibition we selected candidates for sequencing analysis in both cell lines. Sequencing identified inactivating mutations in both alleles of the PARD3 and AS3 genes in the LNCaP and 22RV1 cells, respectively. Introduction of a wild-type PARD3 cDNA into the LNCaP cells resulted in a higher proliferation rate in tissue culture, a higher adhesion of LNCaP cells to the components of extracellular matrix and impaired the growth of the LNCaP cells in soft agar and in a three-dimensional cell-culture.ConclusionThe mutational inactivation in a prostate cancer cell line of the PARD3 gene involved in asymmetric cell division and maintenance of cell-polarity suggests that the loss of cell-polarity contributes to prostate carcinogenesis.
Highlights
Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines
Identifying mutant genes in prostate cancer cell lines using GINI analysis In the original GINI protocol, the mRNA level alterations detected by microarray hybridization after inhibition of nonsense-mediated mRNA decay (NMD) in cells represent the result of two simultaneous processes, i.e., mRNA synthesis and mRNA degradation
The inability to distinguish between mRNA accumulation caused by an induced novel mRNA synthesis and that caused by blocking mRNA degradation is the major source of false-positives produced by GINI analysis
Summary
Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven its usefulness in identifying mutant genes in cancer cell lines. Since triggering the NMD of mutant mRNA requires an initial round of translation, blocking translation with specific drugs, such as emetine, has been shown to abrogate the NMD-mediated degradation of mutant mRNAs [3]. This results in an increased amount of mRNA transcripts from genes containing nonsense or frameshift mutations, which can be detected using gene-expression microarrays. A strategy has been proposed for the identification of genes containing nonsense or frameshift mutations [4] using microarray analysis of mRNA profile alterations resulting from inhibiting NMD in cell lines (GINI)
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