p190B RhoGAP is a negative regulator of the RHO GTPase signaling pathway. Rho signaling is involved in proliferation, migration, polarity and cytoskeletal rearrangement. It has also been shown to be elevated in many forms of cancer. In the developing mouse mammary gland p190B is expressed in the terminal end buds(TEBs), which are the proliferative structures responsible for expanding the ductal system into the mammary fat pad during virgin mammary gland development. Overexpression of p190B during development leads to disruption of the stromal composition of the TEBs. In addition, ductal tree expansion and morphology is abnormal and is characterized by increased branching, lack of organization and stunted expansion of the tree into the fat pad. Overexpression of p190B during pregnancy has also been shown to result in the development of hyperplastic lesions. In this project we aim to determine the cellular and molecular mechanisms by which p190B overexpression disrupts mammary gland development. For this we have mimicked mammary gland ductal development using a three dimensional culture technique. Primary mammary epithelial cells(MECs) are harvested and placed in single cell suspension in Matrigel reconstituted basement membrane(rBM). Individual cells seeded in Matrigel-rBM form hollow acinus-like colonies of cells, which resemble the natural ductal structure formed in vivo in the mammary gland. Primary MECs are obtained from tetracycline regulatable p190B transgenic mice. The MECs harvested from these mice are compared against wild type mice MECs in the 3D culture. Using this method it was determined that p190B overexpression altered proliferation, polarity, cell death rates and morphology.