The G-protein-coupled estrogen receptor (GPER) mediates rapid non-genomic effects of estrogen. Although GPER is able to induce proliferation, it is down-regulated in breast, ovarian and colorectal cancer. During cancer progression, high expression levels of GPER are favorable for patients’ survival. The GPER-specific agonist G1 leads to an inhibition of cell proliferation and an elevated level of intracellular calcium (Ca²⁺). The purpose of this study is to elucidate the mechanism of G1-induced cell death by focusing on the connection between G1-induced Ca²⁺ depletion and endoplasmic reticulum (ER) stress in the estrogen receptor positive breast cancer cell line MCF-7. We found that G1-induced ER Ca²⁺ efflux led to the activation of the unfolded protein response (UPR), indicated by the phosphorylation of IRE1α and PERK and the cleavage of ATF6. The pro-survival UPR signaling was activated via up-regulation of the ER chaperon protein GRP78 and translational attenuation indicated by eIF2-α phosphorylation. However, the accompanying pro-death UPR signaling is profoundly activated and responsible for ER stress-induced cell death. Mechanistically, PERK-phosphorylation-induced JNK-phosphorylation and IRE1α-phosphorylation, which further triggered CAMKII-phosphorylation, are both implicated in G1-induced cell death. Our study indicates that loss of ER Ca²⁺ is responsible for G1-induced cell death via the pro-death UPR signaling.

Inflammation and alterations in essential protein structures in the brain might also change the cellular distribution in the cerebrospinal fluid (CSF).

Alterations in both the expression and function of the non-receptor tyrosine kinase Zap70 are associated with numerous human diseases including immunodeficiency, autoimmunity, and leukemia. Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLCγ1 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70^C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70^C564R, is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70^C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70^C564A displayed the hyperphosphorylation of TCR-ζ and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.

Patients with estrogen receptor positive breast cancer are usually receiving an anti-estrogen therapy by either aromatase inhibitors or selective estrogen receptor mediators such as tamoxifen. Nevertheless, acquired resistance to tamoxifen under treatment frequently hampers therapy. One proposed explanation for this phenomenon is the interaction of the tumor cells with cells of the tumor microenvironment via the Insulin-like growth factor RNA binding protein 5/B-cell lymphoma 3 (IGFBP5/BCL3) axis. Here we investigated whether a high expression of BCL3 either cytoplasmic or nuclear is associated with the occurrence of a relapse under anti-estrogen therapy in patients. Formaldehyde-fixed, paraffin-embedded samples of 180 breast cancer patients were analyzed for BCL3 expression by immunohistochemistry. An immunoreactive score (IRS) was calculated from staining intensity in cytoplasm and nucleus as well as the percentage of positive tumor cells. These scores were correlated with clinico-pathological parameters using cross-tabulation analysis and patients’ relapse free and overall survival by Kaplan–Meier analysis and Cox regression. A tamoxifen-adapted MCF-7 derived cell line was investigated for BCL3 localization by immunofluorescence. The cytosolic BCL3-IRS significantly correlated with the proliferation marker Ki-67, and with the occurrence of a relapse under tamoxifen treatment. Nuclear score correlated only with tamoxifen-relapse. In survival analysis, both scores were highly significant prognostic factors for relapse free, but not for overall survival. This was especially obvious for estrogen receptor positive and HER2/NEU negative cases as well as lobular breast cancer. Tamoxifen-treated, but not aromatase-treated patients had a poor survival when BCL3 scores were high. A tamoxifen adapted cell line exhibited a reduced expression and mainly nuclear localization of BCL3, compared to the parental estrogen receptor positive cell-line MCF-7. Altogether, these data strongly support a function of BCL3 in tamoxifen resistance and its potential use as a predictive biomarker for tamoxifen resistance.

Our knowledge about respiratory virus spreading is mostly based on monolayer cultures that hardly reflect the complex organization of the airway epithelium. Thus, there is a strong demand for biologically relevant models. One possibility to study virus spreading at the cellular level is real-time imaging. In an attempt to visualize virus spreading under somewhat more physiological conditions, Calu-3 cells and human primary fibroblasts were co-cultured submerged or as air-liquid interface (ALI). An influenza A virus (IAV) replicating well in cell culture, and carrying a red fluorescent protein (RFP) reporter gene was used for real-time imaging. Our three-dimensional (3D) models exhibited important characteristics of native airway epithelium including a basement membrane, tight junctions and, in ALI models, strong mucus production. In submerged models, first fluorescence signals appeared between 9 and 12 h post infection (hpi) with a low multiplicity of infection of 0.01. Virus spreading further proceeded in the immediate vicinity of infected cells. In ALI models, RFP was found at 22 hpi and later. Consequently, the progression of infection was delayed, in contrast to the submerged model. With these features, we believe that our 3D airway models can deliver new insights in the spreading of IAV and other respiratory viruses.