CHARACTERIZATION OF THE CELLULAR LOCALIZATION OF BASE

University Of South Dakota, Vermillion SD

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Tumor markers provide information for assessing disease risk, prognosis, metastatic potential and evaluation of response to therapy. Previously, a new breast cancer gene designated BASE (breast cancer and salivary gland expression) has been identified by generating a library that was enriched for genes encoding secreted and membrane proteins. Of 62 normal tissues analyzed, BASE was only expressed in salivary gland. BASE was also expressed in human breast cancers. The deduced amino acid sequence of BASE predicts that it is secreted. BASE is predicted to have a 20 amino acid N-terminal signal sequence, and has homology to other secreted proteins, such as Latherin and the PLUNC family. To further characterize BASE, we need to verify its cellular localization. As a first step, an in vitro expression system using 293T cells and the ZR-75-1 breast cancer cell line will be developed. A plasmid will be constructed containing the BASE ORF cloned 5'of a Myc tag using the vector pcDNA3.1-Myc/His. This expression construct will be transfected into 293T and ZR-76-1 cells. After 48 hours, the culture medium and total cell extracts will be examined for the presence of BASE-Myc by Western immunoblot analysis using a monoclonal antibody against the Myc tag.