Miguel Cuevas and Joseph Wilkerson, Utah Valley University
Biology
Ovarian cancer is the leading cause of death from gynecologic malignancies in the United States and is the fifth leading cause of cancer death among American women. It is estimated that over 22,000 women in 2012 will be diagnosed with ovarian cancer in the United States and approximately 15,500 women will succumb to the disease. This is due to the fact that only 20 percent of cases are diagnosed before the cancer has spread to the peritoneal cavity. Currently, there are no reliable, standard screening tests; the only diagnostic test currently available is the CA125 tumor antigen blood test. This test is inadequate and not available as a general screening tool; additional diagnostics are required to effectively diagnose this disease. It has been previously shown that the tight junction protein Claudin-16, found only in normal kidney, is aberrantly expressed in epithelial ovarian tumors. Therefore, this protein is a good candidate for ovarian cancer diagnostics and targeted therapy. By identifying the promoter region that controls cldn-16 gene expression in ovarian tumors, we can create a luciferase reporter assay to identify cells that express Claudin-16 in culture. To do this, PCR-amplify of various upstream regulatory elements previously identified in kidney cell lines were subcloned into the pGL3 luciferase reporter vector. A higher amount of luminescence is present if the promoter sequence successfully up-regulates the luciferase gene in the vector. This is measured using a Dual Luciferase Assay to determine which promoter region is responsible for the over-expression of Claudin 16. Promoter activity was verified in kidney cell lines that normally express Claudin-16. Next, the assays will be repeated in ovarian cancer cell lines known to express Claudin-16 compared to cell lines that do not express the protein. The promoter assay will then be tested on a collection of ovarian cancer cell lines to determine if luciferase activity correlates with Claudin-16 expression. Once validated, we can test our construct as a cell based assay for identifying therapeutics that can lower Claudin-16 expression in ovarian cancer cells.