The cells were left to grow for 24 h and the medium was replaced with fresh medium or with that containing AP1510 and maintained for a further 48 h. lumen, a single layer of polarized luminal epithelial cells surrounded by myoepithelial cells, and a basement membrane. We have previously shown that human mammary epithelial cells (MECs) form acini-like structures containing a single layer of polarized, growth-arrested cells when grown within a matrix rich in laminin and collagen IV (Matrigel, derived from the EnglebrethCHolm Swarm (EHS) tumour)1,2. The epithelial cells within acini and in culture have an apico-basal distribution of polarity markers such as ZO-1, E-cadherin and 64 integrins. They also deposit collagen IV and secrete sialomucin in their basal and apical surfaces, respectively1,2, indicating that the acinar structures formed in culture closely mimic the acini in an adult breast. Early stages of breast cancer (hyperplasia and ductal carcinoma (DCIS)) are characterized by an increased proliferation of epithelial cells, a loss of acinar organization and filling of the luminal space3. However, a lack of acinar organization and the acquisition of invasive behaviour are later events involved in progression towards malignancy3. Here we show that growth-arrested human mammary epithelial acinar structures can be used to study the early stages of carcinogenesis in culture. The epidermal growth factor (EGF) family of growth factors consists of at least ten different members that bind and activate four receptors, namely ErbB1 (EGF receptor/HER1), ErbB2 (HER2/Neu), ErbB3 and ErbB4. Binding of EGF family ligands to ErbB receptors induces receptor activation by both homodimerization and heterodimerization, thus generating a complex array of combinatorial signals4C6. However, ErbB2 has been shown to be the preferred heterodimerization partner4,7, indicating that ErbB2 has a central role among ErbB receptors. ErbB2 is amplified and overexpressed in 20C80% of DCIS cases, and overexpression of ErbB2 is correlated with a poor clinical prognosis of node-positive tumours7,8. Overexpression of ErbB2 in cultured cells induces both ligand-independent receptor phosphorylation and cellular transformation9C11. Hence, under conditions in which ErbB2 is highly amplified relative to other family members kinase with MBP as the substrate. The kinase assay blot was reprobed with anti-Erk2 antibodies to determine the levels of Erk2 protein in kinase reactions. To determine whether the homodimerization of p75.B1 and p75.B2 can activate downstream signalling pathways in this cell system, we analysed the activation of the mitogen-activated protein kinase Erk2. As observed in Rat1 fibroblasts14, homodimerization and activation of either p75.B1 or p75.B2 resulted in a 5C7-fold increase in Erk2 kinase activity (Fig. 1c), indicating that both homodimers were equally competent to initiate the signalling cascade required for the activation of Erk2. The Erk2 kinase activity stimulated by AP1510 was 2C4-fold weaker than that stimulated by 8 nM EGF (Fig. 1c; CNA1 compare lanes Ezatiostat 2 and 3 with lane 4, and lanes 6 and 7 with lane 8). Activation of both p75.B1 and p75.B2 homodimers induced a modest increase in Ser 473 phosphorylation of Akt, a downstream target of the PtdIns-3-OH kinase signalling pathway (data not shown). To evaluate whether the dimerization of chimaeric ErbB receptors can stimulate a biological response, we evaluated the ability of ErbB homodimers to induce proliferation in the absence of EGF when cells are grown on a plastic tissue culture plate (two-dimensional (2D)). Like the parental MCF-10A cells, neither the p75.B1-expressing cells nor the p75.B2-expressing cells were able to proliferate in the absence of exogenous EGF (Fig. 2a). Activation of either p75.B1 or p75.B2 homodimers was sufficient to stimulate EGF-independent proliferation; however, p75.B2 homodimers were more potent than p75.B1 homodimers (Fig. 2a). These results indicate that dimerization of p75.B1 or p75.B2 chimaeras is sufficient to stimulate a biological response. Open in a separate window Figure 2 MCF-10A cells form growth arrested polarized acinar structures in Matrigela, Cells expressing different ErbB chimaeras were plated Ezatiostat either in the presence of EGF or AP1510. Cell numbers were determined after 5 days. The graph represents an average of three experiments. b, Morphology of acinar structures formed by MCF-10A cells plated in Matrigel for 12 days. Phase image of Ezatiostat an acinus at higher magnification is shown in the right inset. The acini were labelled with DAPI and a confocal image through the middle.