Doctor of Philosophy (PhD)
Dissociable Antibody Microarray (DAMA) staining is a novel technique that integrates protein microarrays with conventional immunostaining techniques. It can simultaneously determine the expression and subcellular localizations (SCLs) of hundreds of proteins in cultured cells. I optimized this technology for protein expression and SCL profiling, and generated expression profile data analysis program DAMAPEP, molecular image database management program ChipView and automatic SCL assignment program DAMASCL. We demonstrated the application of this technique in the identification of potential biomarkers for breast cancer. We compared the expression profiles of 312 proteins among ten breast cell lines and identified 10 differentially expressed proteins. Among those proteins, RAIDD, Rb p107, Rb p130, SRF and Tyk2 were confirmed by western blot and statistical analysis to have higher expression levels in cancer breast cells than in normal breast cells. We also compared the SCL profiles of 325 proteins among nine breast cell lines, and identified one protein, Cyclin B1, with different SCLs between two normal and seven cancer breast cell lines. With individual immunostaining, Cyclin B1 was confirmed to localize in the cytoplasm of seven cancer cells and in both cytoplasm and nuclei of two normal cells and to have higher expression levels in the seven cancer cell lines. We expanded the scale of DAMA staining to include 400 antibodies per array and surveyed SCL profiles of 400 antibodies in five prostate cell lines. Five proteins were identified to have altered SCL patterns between normal and cancer prostate cell lines. GRK2 was so far confirmed to localize ubiquitously in the cytosol of three normal and one cancer prostate cell lines while concentrating at certain regions right beneath plasma membrane in the other two cancer prostate cell lines. We also extended the application of DAMA staining to interrogate protein expression profiles in tissue samples and found 3 proteins with differential expression in two tissue samples from different breast cancer patients, demonstrating the potential use of DAMA staining in carcinoma characterization and classification. Database of annotated protein molecular images obtained from DAMA staining need to be created and shared for better understanding of cancer biology.
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Fu, Guanyuan, "Development and application of Dissociable Antibody Microarray (DAMA) staining technique" (2009). LSU Doctoral Dissertations. 3432.