High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy

SummaryCostimulatory molecules are important regulators of T cell activation and thus favored targets for therapeutic manipulation of immune responses. One of the key costimulatory receptors is CD80, which binds the T cell ligands, CD28, and CTLA-4. We describe a set of small compounds that bind with high specificity and low nanomolar affinity to CD80. The compounds have relatively slow off-rates and block both CD28 and CTLA-4 binding, implying that they occlude the shared ligand binding site. The compounds inhibit proinflammatory cytokine release in T cell assays with submicromolar potency, and as such, they represent promising leads for the development of novel therapeutics for immune-mediated inflammatory disease. Our results also suggest that other predominantly β proteins, such as those that dominate the cell surface, may also be accessible as potentially therapeutic targets

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells

Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

<p>Abstract</p> <p>Background</p> <p>Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs).</p> <p>Findings</p> <p>Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.</p> <p>To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment.</p> <p>Conclusion</p> <p>In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb-/- MEFs could be identified. These data and the analysis provide a starting point for further experimental studies to identify target genes for pharmacological intervention and ultimately remodeling of white adipose tissue into brown adipose tissue.</p

Functional Interactions between Retinoblastoma and c-MYC in a Mouse Model of Hepatocellular Carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes

Claudin 13, a Member of the Claudin Family Regulated in Mouse Stress Induced Erythropoiesis

Mammals are able to rapidly produce red blood cells in response to stress. The molecular pathways used in this process are important in understanding responses to anaemia in multiple biological settings. Here we characterise the novel gene Claudin 13 (Cldn13), a member of the Claudin family of tight junction proteins using RNA expression, microarray and phylogenetic analysis. We present evidence that Cldn13 appears to be co-ordinately regulated as part of a stress induced erythropoiesis pathway and is a mouse-specific gene mainly expressed in tissues associated with haematopoietic function. CLDN13 phylogenetically groups with its genomic neighbour CLDN4, a conserved tight junction protein with a putative role in epithelial to mesenchymal transition, suggesting a recent duplication event. Mechanisms of mammalian stress erythropoiesis are of importance in anaemic responses and expression microarray analyses demonstrate that Cldn13 is the most abundant Claudin in spleen from mice infected with Trypanosoma congolense. In mice prone to anaemia (C57BL/6), its expression is reduced compared to strains which display a less severe anaemic response (A/J and BALB/c) and is differentially regulated in spleen during disease progression. Genes clustering with Cldn13 on microarrays are key regulators of erythropoiesis (Tal1, Trim10, E2f2), erythrocyte membrane proteins (Rhd and Gypa), associated with red cell volume (Tmcc2) and indirectly associated with erythropoietic pathways (Cdca8, Cdkn2d, Cenpk). Relationships between genes appearing co-ordinately regulated with Cldn13 post-infection suggest new insights into the molecular regulation and pathways involved in stress induced erythropoiesis and suggest a novel, previously unreported role for claudins in correct cell polarisation and protein partitioning prior to erythroblast enucleation

3D Molecular Simulation on the Web

[中文文摘]绍几种基于Web上的三维分子结构模拟技术:VRML及Chime、Chem3D、HyperChem插件,并讨论它们的优缺点.这些模拟技术具有可操作的特点,可以进行旋转、缩放、平移等实时交互操作,并改变其形状和颜色的表现形式.三维分子结构模拟可以增强化学网络课程的表现能力,提高教学效果,是发展远程教育的重要技术.[英文文摘]Several techniques of 3D molecular simulation based on Web page were introduced, which are VRML, Chime, Chem3D and HyperChem plugins. Their advantages and shortcomings were also discussed.The rotations, zooms, transformations can be operated interactively, and several color and display modes are also available. 3D molecular simulations can improve performances of chemical network courses and enhance educational effects. They are important techniques on developing the long distance educations