CLDN-16, -4, and -3. Clone tail sequences using. Expresso T7 Cloning System. (Lucigen Corp., Middleton, WI). PCR products have been confirmed through gel ...
Expression of the C-terminal domains of the tight junction proteins Claudin-16, -3, and -4 to identify interacting proteins in epithelial ovarian carcinoma Brandon Davies, Colleen Hough Department of Biology/Biotechnology, College of Science and Health Utah Valley University
ABSTRACT The Claudin family of proteins are a main component of tight junctions in the upper region of epithelial cells that act as gateways for the exchange of water and solutes while also helping determine the cell’s polarity and function. Changes in these proteins cause changes in phenotype and function of normal epithelial cells, such as proliferation control, transepithelial resistance, polarity, and solute transport. Claudin-16 is often aberrantly expressed in breast and ovarian cancer, while Claudins 3 and 4 are highly overexpressed in epithelial ovarian carcinoma (EOC). The location of these proteins is also correlated with oncogenic transformations and cell proliferation. We are currently cloning the C-termini of Claudins -16, -4, and -3, which contain a known PDZ-binding motif and may interact with other junction proteins or with proteins involved in interesting signaling pathways. Identifying theses interacting proteins through pull down assays may provide insights into the pathogenesis of ovarian cancer.
METHODS Obtain and identify C-terminal, cytoplasmic tail sequences for CLDN-16, -4, and -3. Clone tail sequences using Expresso T7 Cloning System (Lucigen Corp., Middleton, WI).
CONCLUSIONS Future processes include using the His affinity tag to capture proteins bound to Claudin tails by pull down assay to be analyzed by SDS-PAGE and, ultimately, the corresponding genes cloned and sequenced. Pull down assays can be completed using FPLC or His-tag affinity to metal [cobalt (Co2+) or nickel (Ni2+)] beads.
INTRODUCTION EOC is the sixth most common cancer in US women. The longterm cure rates are low due to the lack of reliable biomarkers for early disease detection, resulting in advanced stage diagnosis. Approximately 75%-80% of ovarian cancers are diagnosed at stages II-IV with a 10% 5-year survival rate despite aggressive treatments. Claudin proteins are being studied as possible biomarkers as they are aberrantly overexpressed in EOC tumors.
This study can potentially provide crucial information in relation to how members of the Claudin family interact with other proteins that are commonly found in tissues that are misregulated in cancer. With this data treatments can be improved to increase the responsiveness of ovarian cancer patients. Temperature gradient PCR to determine precise reaction conditions for maximum product quantity.
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Determining the specific characteristics of these Claudin proteins can prove to be of incredible benefit in cancer treatments. As these proteins are targeted during these therapies, these tight junctions may then send normal signals, which in turn can regulate the cell normally.
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Design and carry out protein pull down assays using His-tag affinity to metal [cobalt (Co2+) or nickel (Ni2+)] beads. Elute with imidazole. Use antibodies with known affiliations for observation through SDS-PAGE.
RESULTS PCR products have been confirmed through gel electrophoresis. Continuation will include cloning and expression of the Cterminal tails and protein pull down assays to determine the specific binding characteristics with other interacting proteins.
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ACKNOWLEDGEMENTS We would like to thank Utah Valley University, Salt Lake Community College, Dr. Colleen Hough, Dr. Marie Balzotti, and Dr. Mark Bracken for their contributions to this project. Additional appreciation to Dixie State University and the Utah Conference for Undergraduate Research for the chance to present.
