ABSTRACT
INTRODUCTION: Breast cancer, the most common malignancy in women, still holds many secrets. The causes for non-hereditary breast cancer are still unknown. To elucidate any role for circulating naturally secreted proteins, a screen of secreted proteins' influence of MCF10A cell anchorage independent growth was set up. METHODS: To systematically screen secreted proteins for their capacity to transform mammalian breast epithelial cells, a soft agar screen of MCF10A cells was performed using a library of ~ 470 secreted proteins. A high concentration of infecting viral particles was used to obtain multiple infections in individual cells to specifically study the combined effect of multiple secreted proteins. RESULTS: Several known breast cancer factors, such as Wnt, FGF and IL were retained, as well as factors that were previously unknown to have a role in breast cancer, such as paraoxonase 1 and fibroblast growth factor binding protein 2. Additionally, a combinatory role of Interleukin 6 with other factors in MCF10A anchorage-independent growth is demonstrated. CONCLUSION: The transforming effect of combinations of IL6 with other secreted proteins allows studying the transformation of mammary epithelial cells in vitro, and may also have implications in in vivo studies where secreted proteins are upregulated or overexpressed.
ABSTRACT
Multivalent binding of glycans on pathogens and on mammalian cells by the receptors DC-SIGN (CD209) and DC-SIGNR (L-SIGN, CD299) is dependent on correct disposition of the C-type carbohydrate-recognition domains projected at the C-terminal ends of necks at the cell surface. In the work reported here, neck domains of DC-SIGN and DC-SIGNR expressed in isolation are shown to form tetramers in the absence of the CRDs. Stability analysis indicates that interactions between the neck domains account fully for the stability of the tetrameric extracellular portions of the receptors. The neck domains are approximately 40% alpha-helical based on circular dichroism analysis. However, in contrast to other glycan-binding receptors in which fully helical neck regions are intimately associated with C-terminal C-type CRDs, the neck domains in DC-SIGN and DC-SIGNR act as autonomous tetramerization domains and the neck domains and CRDs are organized independently. Neck domains from polymorphic forms of DC-SIGNR that lack some of the repeat sequences show modestly reduced stability, but differences near the C-terminal end of the neck domains lead to significantly enhanced stability of DC-SIGNR tetramers compared to DC-SIGN.
