EXPRESSION AND FUNCTION OF CUL 5/VACM 1 IN BREAST CANCER

Midwestern University, Downers Grove IL

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Abstract

The overall goal of this project is to gain insight into the development of breast cancer through a better understanding of the cellular function(s) of the putative tumor suppressor gene cul-5/VACM-1. The hypothesis to be tested is that CUL5 acts as a tumor suppressor in breast cancer and is involved in cell growth and cell cycle events by participating in the ubiquitin-mediated degradation of cell cycle regulatory proteins. This hypothesis will be tested through the following three specific aims: (1) Expression of CUL5 mRNA and protein will be evaluated in breast epithelial cells/normal breast tissues vs breast cancer cell lines/biopsy specimens using RTPCR, Northern and Western blot and immunochemistry. (2) The influence of CUL5 on cell growth and cell cycle events will be evaluated by transiently transfecting a breast epithelial cell line (MCF10A) and breast cancer cell lines (MCF7, MDAMB231) with CUL5 expression construct. Proliferation will be determined with 3H-thymidine and BrdU and cell cycle events monitored by propidium iodide staining and flow cytometry. (3) To evaluate if CUL5 is involved in the ubiquitin-mediated degradation of cell cycle regulatory proteins, cells will be transiently transfected with a CUL5 expression construct, and the expression and ubiquitination of key short-lived cell cycle regulatory proteins will be determined using immunoprecipitation and Western blot.