4501

Objective: Nonsense-mediated decay (NMD) is a normal protective cellular mechanism by which mRNA transcripts containing premature termination codons are rapidly degraded before the truncated proteins can be translated. Recently, a novel technique, called Gene Identification by Nonsense-mediated Inhibition (GINI), has been developed which pharmacologically inhibits NMD to allow mutated transcripts to accumulate. GINI has been successfully utilized to detect mutations in prostate and colon cancer. To date, this technique has not been utilized in gynecologic malignancies. The objective of this study is to use this newly-developed GINI technology to detect novel mutations in human epithelial ovarian cancer cells that may be important in ovarian cancer tumorigenesis. Methods: Five ovarian cancer cell lines and 1 culture of normal human ovarian surface epithelial cells (HOSE) were grown to 60-70% confluence in the usual manner. Cell lines were then treated with the translation-inhibiting drug, Emetine, at a concentration of 100 ug/ml. Actinomycin D was used at a concentration of 2ug/ml to prevent the upregulation of stress-response genes. Cells were treated with both drugs for 4 hours. Additional plates of each cell line were also treated with the drug diluents, PBS and DMSO, as negative controls. The cells were then lysed and RNA extracted. RNA was reversed transcribed, labeled with biotin by in vitro transcription, fragmented and hybridized to Affymetrix U133 plus 2 Genechips. Expression values for the over 54,000 transcripts represented by the probe sets on the Genechip were estimated by GC-RMA normalization algorithm. The ratios between emetine treated and controls were calculated for each cell line as well as the HOSE. Genes that are up-regulated by emetine treatment in cancer cell lines but not in the HOSE were selected by the OmniViz data mining tool. Results: We have generated 12 expression profiles for 5 ovarian cancer cell lines and 1 culture of HOSE (for both control and emetine treated experiments). After subtracting any genes that are up-regulated by emetine treatment in the HOSE culture, we have identified from 95 to 708 genes that are up-regulated in each of the five cell lines. Among these genes, thirty-five up-regulated genes were shared by two different cell lines. These genes are involved in lipid metabolism, regulation of transcription, proteolysis/peptidolysis, and signal transduction. Conclusion: We have identified genes with potential mutations in several ovarian cancer cell lines using the GINI strategy. Sequencing analyses are in progress to validate and determine the site of the nonsense mutations in these identified genes.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]