Syk Loss in Breast Cells described [12,13]. However, homozygous deletion of Syk is perinatal lethal, due to failure of lymphatic and blood vessels to properly separate [14]. Other aspects of development such as formation of mammary gland were not reported. Here we determine the role of Syk in normal epithelium using these in vitro and in vivo models. Taken together our results are consistent with a critical role for Syk in blocking pre-neoplastic cell proliferation and invasion and suggest that Syk does so by affecting several major signaling pathways important for normal breast development and maintenance. Results In vitro studies Knockdown of Syk induces increased migration and cell proliferation in human breast epithelial cells. First, we investigated whether Syk loss in normal human breast epithelial cells would have an impact on the ability of these cells to proliferate and invade. For these studies, we utilized non- transformed, immortalized MCF10A human breast epithelial cells (MCF10A1) and two cell lines derived from them, MCF10AneoT, and MCF10DCIS.com (DCIS.com), with characteristics of normal, hyperplastic, and ductal carcinoma in situ (DCIS), respectively. MCF10A contained the greatest amount of Syk and MCF10AneoT and DCIS.com each had progressively less (Figure 1A). Syk siRNA treatment effectively lowered Syk protein levels up to 90% in all three cell lines (Figure 1A). Analysis of cell proliferation and growth revealed that loss of Syk in each cell line led to enhanced cell proliferation, approximately 4-fold greater in MCF10A cells, 2 fold greater in MCF10AneoT, and 0.4 greater in DCIS.com (Figures 1B and 1C). Next, we asked whether normal cells or the transformed MCF10A derivatives migrated more efficiently following Syk knockdown using a scratch/wound assay. Cell velocities and rate of gap closure of the monolayer were significantly increased in each cell line following Syk siRNA knockdown (Figures 1D and Figure 1. Cell proliferation and migration of MCF10A, MCF10AneoT, and DCIS.com human mammary epithelial cells. (A) Decreases in Syk protein levels are shown for control versus Syk siRNA transfection of normal MCF10A cells and tumorigenic MCF10AneoT and DCIS.com cells. Increased cell proliferation (B) and cell growth (C) was observed following Syk knockdown. (D) Increased velocity of MCF10A, MCF10AneoT, and DCIS.com cell migration was observed in Syk knockdown cells using a scratch/wound assay. (B–D) Data is represented as means +/2 SEM. P values are significant by the student’s t-test for all cell lines, average of 3 independent experiments. doi:10.1371/journal.pone.0007445.g001 PLoS ONE | www.plosone.org 2 October 2009 | Volume 4 | Issue 10 | e7445