Transcript and protein profiling identifies signaling, growth arrest, apoptosis, and NF-κB survival signatures following GNRH receptor activation

GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60) in vitro and in vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene, CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G(2)/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60 in vitro, and p-NF-κB and IκBϵ were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation

Isolation and Characterization of a Metastatic Hybrid Cell Line Generated by ER Negative and ER Positive Breast Cancer Cells in Mouse Bone Marrow

BACKGROUND: The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior. METHODS: ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC. RESULTS: The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines. CONCLUSIONS: Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity

Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered

Influenza-Specific T Cells from Older People Are Enriched in the Late Effector Subset and Their Presence Inversely Correlates with Vaccine Response

T cells specific for persistent pathogens accumulate with age and express markers of immune senescence. In contrast, much less is known about the state of T cell memory for acutely infecting pathogens. Here we examined T cell responses to influenza in human peripheral blood mononuclear cells from older (>64) and younger (<40) donors using whole virus restimulation with influenza A (A/PR8/34) ex vivo. Although most donors had pre-existing influenza reactive T cells as measured by IFNγ production, older donors had smaller populations of influenza-responsive T cells than young controls and had lost a significant proportion of their CD45RA-negative functional memory population. Despite this apparent dysfunction in a proportion of the older T cells, both old and young donors' T cells from 2008 could respond to A/California/07/2009 ex vivo. For HLA-A2+ donors, MHC tetramer staining showed that a higher proportion of influenza-specific memory CD8 T cells from the 65+ group co-express the markers killer cell lectin-like receptor G1 (KLRG1) and CD57 compared to their younger counterparts. These markers have previously been associated with a late differentiation state or immune senescence. Thus, memory CD8 T cells to an acutely infecting pathogen show signs of advanced differentiation and functional deterioration with age. There was a significant negative correlation between the frequency of KLRG1+CD57+ influenza M1-specific CD8 T cells pre-vaccination and the ability to make antibodies in response to vaccination with seasonal trivalent inactivated vaccine, whereas no such trend was observed when the total CD8+KLRG1+CD57+ population was analyzed. These results suggest that the state of the influenza-specific memory CD8 T cells may be a predictive indicator of a vaccine responsive healthy immune system in old age

Stressful situation if CENP-A not front and CENter

The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation

Standardization and validation of assays determining cellular immune responses against influenza.

International audienceInfluenza vaccine efficacy does not always correlate with humoral immune responses. Recent reports indicate that the cellular immune response also contributes to protection, however robust assays are lacking. We standardized and validated assays for detection of human influenza-specific cellular responses in four international laboratories. The production of granzyme B as marker of T cell-mediated cytotoxicity and release of Th1 and Th2 cytokines were evaluated. The granzyme B and cytokine assays were specific, accurate, precise, and robust. Replicate stimulations with PBMC from the same donors showed an intra-laboratory robustness (coefficient of variation) for quantitation of granzyme B of 33% and for cytokines - including IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4, IL-13, GM-CSF and including the log IFN-gamma/IL-10 ratio - of 52%. The inter-laboratory robustness for detection of granzyme B was 29% and for detection of all cytokines was 49%. The assays can now be used for determining cell-mediated immunity and explored as correlates of protection. Moreover, the precision and robustness of these cellular assays allow the reliable detection of cellular responses even in small study populations

GLA-SE, a Synthetic Toll-like Receptor 4 Agonist, Enhances T-Cell Responses to Influenza Vaccine in Older Adults

Background. The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza. Methods. The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant–stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection. Results. GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor α, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon γ to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults. Conclusions. Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults