Date of Award

8-1-2004

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Abstract

The BOG25 family proteins, BOG25 and 7a5, contain domains of the EH- Network family of endocytosis, intracellular sorting and cell fate determination proteins as well as a C-terminal death domain that may play an important role in apoptosis. No other known proteins contain the same domain combinations which makes this family unique. The normal physiological roles of the BOG25 family of proteins are currently unknown, and this is the first study of the possible role of BOG25 and 7a5 in cancer. Additionally, this study is the first to confirm the transcription of mRNA for 7a5, the putative protein identified as similar to BOG25 by the Human Genome Project. Three cDNA sequences were subcloned, sequenced and aligned with 100% identity, the predicted coding region of 7a5 from the genomic sequence submitted by the Human Genome Project.

The hypothesis of this study is that BOG25 and 7a5 proteins play an important role in the cancer phenotype, and therefore, will have a substantial differential expression between the cancer cell lines and/or between the cancer and normal cell lines. Western blot, subcellular fractionation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were used to analyze three sets of cell lines: bladder, prostate and breast. The most advanced cancerous bladder cell line, HTB 5, expressed a very high level of 7a5 protein, while the most differentiated cancer cell line, HTB 2, expressed the lowest level. Conversely, BOG25 and 7a5 proteins in prostate cell lines showed no major difference between the cell lines. However, the breast cancer cell line, Hs578T expressed BOG25 protein at a high level compared to all of the other breast cell lines. Based on these results, the breast cell lines were further characterized using subcellular fractionation and RT-PCR to examine mRNA and corresponding protein levels. Subcellular fractionation revealed BOG25 expression at approximately a 1:1 ratio of PNF to NF in all breast cell lines including Hs578T cells while 7a5 was expressed at higher levels in the PNF than NF in all cell lines. No major alteration in subcellular localization was observed for either BOG25 or 7a5 in the Hs578T cell line compared to the other breast cell lines. RT- PCR of triplicate total RNA samples from breast cells showed that Hs578T expressed a very high level of BOG25 mRNA compared to all other cell lines. Corresponding triplet lysates analyzed for BOG25 and 7a5 protein expression showed that protein levels were comparable to the mRNA levels where all cell lines expressed BOG25 at a much lower level than Hs578T. Only one sample showed a correctly sized 7a5 mRNA band for the MDA cell line. All samples for the other cell lines expressed similar 7a5 mRNA levels. Triplicate 7a5 protein data showed subtle differences among cell lines that were comparable to the mRNA data. The unique domain combinations and the highest expression in the most advanced cancer are persuasive reasons for BOG25 and 7a5 to be examined further as prognostic markers in breast and bladder cancer tissues, respectively

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