Understanding of complex protein interactions with respect to anchorage independence.

Anchorage-independent growth of cells in soft agar is one of the hallmark characteristics of cellular transformation and uncontrolled cell growth. It may be considered as one of the most stringent assays for detecting malignant transformation of cells. Here, we describe a retroviral infection of a library of small secretory proteins and the use of the soft agar assay to obtain and study novel interacting protein combinations that cause cell transformation.

Systematic analysis of secreted proteins reveals synergism between IL6 and other proteins in soft agar growth of MCF10A cells.

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.

LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation.

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-kappaB (NF-kappaB). Although LPS-induced activation of NF-kappaB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-kappaB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-kappaB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-kappaB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-kappaB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKbeta. NF-kappaB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-kappaB activity in the liver, thereby partially protecting mice against LPS/D-(+)-galactosamine-induced mortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-kappaB activation.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-kappaB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-kappaB also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-kappaB inhibitors in the liver. This dual function of NF-kappaB emphasizes the need for therapeutics that can inhibit both TNF-induced NF-kappaB activation and cell death. Here we describe that adenoviral expression of the NF-kappaB inhibitory protein ABIN-1, but not an IkappaBalpha superrepressor (IkappaBalpha(s)), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IkappaBalpha(s) suggest a role for an NF-kappaB independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-kappaB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-kappaB inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.

Yeast two-hybrid screening for proteins interacting with the anti-apoptotic protein A20.

The yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors. Therefore, the protein of interest (bait) is fused to a DNA-binding domain, and complimentary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined and transcription from a reporter gene is started. Here, we will give a comprehensive guide for the GAL4-based two-hybrid system, exemplified by the detection of binding partners for the zinc finger protein A20. The latter is an inducible cellular inhibitor of tumor necrosis factor (TNF)-induced apoptosis and nuclear factor (NF)-kappaB-dependent gene expression. Yeast two-hybrid screening with A20 as bait revealed several A20-binding proteins, including A20 itself, members of the 14-3-3 family, as well as three novel proteins ABIN-1, ABIN-2, and TXBP151. The latter protein was subsequently shown to mediate at least part of the anti-apoptotic activities of A20, whereas ABIN-1 and -2 are more likely to be involved in the NF-kappaB inhibitory effects of A20.