Abstract
Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.
Highlights
Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma
In this study we compared the proteome of OSE cell lines derived from women with strong histories of familial breast and/or ovarian cancer and mutations in the breast cancer antigen-1 (BRCA1) gene to the proteome of OSE cell lines derived from women without such histories and known mutations
The use of 2-D SDS-PAGE and computer-assisted gel analysis coupled with in-gel digestion and mass spectrometry permitted the isolation and identification of several proteins whose expression was repeatedly altered in family history (FH)-OSE cell lines
Summary
OSE Cell Lines and Culture Conditions—OSE cell lines, originally derived from the ovarian surface epithelial cells from women with and without strong family histories of breast and ovarian cancer [13] at the Fox Chase Cancer Center, were immortalized by transformation with SV-40 large T antigen as described previously [14]. Silver-stained gels were examined visually and scanned using an Image Acquisition Sharp JX330 scanner (Sharp Electronics Corp., Mahwah, NJ) interfaced to a SUN Sparc 4 computer (Sun Microsystems, Mountain View, CA). Images of the gels and an OSE data base was created using BioImage 2-D Analyzer software (Genomic Solutions, Inc.) on the SUN work station. Protein spots were excised from silver-stained gels and minced with a scalpel, and the gel pieces were washed with 25 mM NH4HCO3 in 50% ACN, dried in a Speedvac, rehydrated in 25 mM NH4HCO3 solution containing trypsin (10 ng/ml, Promega, Madison, WI), and digested overnight at 37 °C. Some peptides were sequenced manually, and those fragment ions were submitted for data base searching using MASCOT and MS-Tag for protein identification (Ref. 21, prospector.ucsf.edu). The data were analyzed with Wholeband Analyzer BioImage software system (Genomic Solutions, Inc.)
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