5285

20-30% of ovarian epithelial carcinomas (OEC) exhibit increased copy numbers of the 20q13 locus, which is the site of several putative oncogenes, including EEF1A2. The roles of these genes in ovarian carcinogenesis are not fully defined. EEF1A2 regulates ribosomal polypeptide elongation, but has also been reported to be antiapoptotic and to interact with the cytoskeleton. EEF1A2 is highly expressed in about 30% of OEC, and enhances neoplastic properties of NIH3T3 cells and of ovarian carcinoma cells (Anand et al., 2002, Nat. Genet. 31:301). We investigated the effects of EEF1A2 overexpression on the phenotype of nontumorigenic human ovarian surface epithelial cells (OSE), the precursor of OEC. A lentiviral construct containing the EEF1A2 gene with a C-terminal V5 tag was introduced into the SV40 Tag/tag expressing nontumorigenic OSE line IOSE-144, generating 13 separate stable EEF1A2-expressing IOSE-144 lines (lines I-144LE(1) to I-144LE(13)). RT-PCR indicated the existence of the EEF1A2-V5 gene in all the I-144LE lines, while western blots demonstrated increased EEF1A2 protein levels. Crisis and senescence of the I-144LE lines were delayed by several passages compared to IOSE-144. I-144LE cells were more serum-independent than the parent line and proliferated even in the complete absence of serum, suggesting autocrine growth factor production. Proliferation assays demonstrated no change in growth rates but increased saturation densities ranging from 10% to 33%. Anchorage independence assays showed that I-144LE lines formed 6 to 7 times more cell colonies as compared with either the IOSE-144 parent line or the β-Gal control line. Finally, DNA fragmentation- and caspase-3 activity assays showed significantly reduced camptothecin-induced apoptosis in I-144LE cells, while siRNA to EEF1A2 greatly enhanced apoptosis compared to controls. The results of this study demonstrate that EEF1A2 overexpression contributes neoplastic characteristics to nontumorigenic precursor cells of OEC and support previous evidence pointing to an oncogenic role of this protein in ovarian carcinogenesis. Supported by NCIC, Canada.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA