Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

The integrin antagonist cilengitide activates alphaVbeta3, disrupts VE-cadherin localization at cell junctions and enhances permeability in endothelial cells.

Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface alphaVbeta3, stimulated phosphorylation of FAK (Y(397) and Y(576/577)), Src (S(418)) and VE-cadherin (Y(658) and Y(731)), redistributed alphaVbeta3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density beta1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of alphaVbeta3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.

Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Nervous glucose sensing regulates postnatal β cell proliferation and glucose homeostasis.

How glucose sensing by the nervous system impacts the regulation of β cell mass and function during postnatal development and throughout adulthood is incompletely understood. Here, we studied mice with inactivation of glucose transporter 2 (Glut2) in the nervous system (NG2KO mice). These mice displayed normal energy homeostasis but developed late-onset glucose intolerance due to reduced insulin secretion, which was precipitated by high-fat diet feeding. The β cell mass of adult NG2KO mice was reduced compared with that of WT mice due to lower β cell proliferation rates in NG2KO mice during the early postnatal period. The difference in proliferation between NG2KO and control islets was abolished by ganglionic blockade or by weaning the mice on a carbohydrate-free diet. In adult NG2KO mice, first-phase insulin secretion was lost, and these glucose-intolerant mice developed impaired glucagon secretion when fed a high-fat diet. Electrophysiological recordings showed reduced parasympathetic nerve activity in the basal state and no stimulation by glucose. Furthermore, sympathetic activity was also insensitive to glucose. Collectively, our data show that GLUT2-dependent control of parasympathetic activity defines a nervous system/endocrine pancreas axis that is critical for β cell mass establishment in the postnatal period and for long-term maintenance of β cell function.

LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.

Tumour immunologists strive to develop efficient tumour vaccination and adoptive transfer therapies that enlarge the pool of tumour-specific and -reactive effector T-cells in vivo. To assess the efficiency of the various strategies, ex vivo assays are needed for the longitudinal monitoring of the patient's specific immune responses providing both quantitative and qualitative data. In particular, since tumour cell cytolysis is the end goal of tumour immunotherapy, routine immune monitoring protocols need to include a read-out for the cytolytic efficiency of Ag-specific cells. We propose to combine current immune monitoring techniques in a highly sensitive and reproducible multi-parametric flow cytometry based cytotoxicity assay that has been optimised to require low numbers of Ag-specific T-cells. The possibility of re-analysing those T-cells that have undergone lytic activity is illustrated by the concomitant detection of CD107a upregulation on the surface of degranulated T-cells. To date, the LiveCount Assay provides the only possibility of assessing the ex vivo cytolytic activity of low-frequency Ag-specific cytotoxic T-lymphocytes from patient material.

Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris

The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.

Multifunctional roles for MALT1 in T-cell activation

The activation of T cells is vital to the successful elimination of pathogens, but can also have a deleterious role in autoimmunity and transplant rejection. Various signalling pathways are triggered by the T-cell receptor; these have key roles in the control of the T-cell response and represent interesting targets for therapeutic immunomodulation. Recent findings define MALT1 (mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1) as a protein with proteolytic activity that controls T-cell activation by regulating key molecules in T-cell-receptor-induced signalling pathways

Dissociation from BiP and Retrotranslocation of Unassembled Immunoglobulin Light Chains Are Tightly Coupled to Proteasome Activity

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (KNS1) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (Knittler et al., 1995). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of KNS1, arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of KNS1 upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of KNS1and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-KNS1complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.

Detection of an asymptomatic right-ventricle cardiac metastasis from a small-cell lung cancer by F-18-FDG PET/CT.

A right heart metastasis of a small-cell lung cancer was found on the whole-body F-fluoro-deoxy-glucose positron emission tomography/computed tomography (F-FDG-PET/CT) of a 69-year-old smoker investigated for a right pulmonary mass discovered on chest radiography after a fracture of the right humerus. The PET scan showed an increased FDG uptake by the mass in the right lung and an intense, atypical focal activity of the right ventricle strongly suggestive of a neoplastic process. CT-guided lung biopsy revealed a small-cell lung cancer and myocardial biopsy confirmed the presence of a cardiac metastasis. The patient was treated with six cycles of chemotherapy followed by radiation therapy, which included the heart lesion. At follow-up PET/CT 2 months after the end of treatment, the abnormal cardiac uptake had disappeared, whereas increased FDG uptake persisted in the pulmonary residual mass.

Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins.

Adrenoceptors are prototypic members of the superfamily of seven transmembrane domain, G protein-coupled receptors. Study of the properties of several mutationally activated adrenoceptors is deepening understanding of the normal functioning of this ubiquitous class of receptors. The new findings suggest an expansion of the classical ternary complex model of receptor action to include an explicit isomerization of the receptors from an inactive to an active state which couples to the G protein ('allosteric ternary complex model'). This isomerization involves conformational changes which may occur spontaneously, or be induced by agonists or appropriate mutations which abrogate the normal 'constraining' function of the receptor, allowing it to 'relax' into the active conformation. Robert Lefkowitz and colleagues discuss the physiological and pathophysiological implications of these new insights into regulation of receptor activity.

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells

Introduction: Cancer stem cells (CSC) display plasticity and self renewal properties reminiscent of normal tissue stem cells but the events responsible for their emergence remain obscure. We have recently identified CSC in Ewing sarcoma family tumors (ESFT) and shown that they arise from mesenchymal stem cells from the bone marrow. Objective of the study: To analyze the mechanisms underlying cancer stem cell development in ESFT. Methods: Primary human mesenchymal stem cells (MSC) isolation from adult and pediatric bone marrow. Retroviral delivery of fusion protein (EWS-FLI1) to primary MSC, and transcriptional and phenotypical analysis. Results: We show that the EWS-FLI-1 fusion gene, associated wit 85-90% of ESFT and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2 and NANOG in human pediatric MSC (hpMSC) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSC expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWSFLI- 1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Conclusion: Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a cancer stem cell phenotype.

Conformational changes modulate the activity of human RAD51 protein.

Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homologue, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity.

Fluorodeoxyuridine mediated cell cycle synchronization in S-phase increases the Auger radiation cell killing with 125I-iododeoxyuridine.

Aim: 125I-iododeoxyuridine is a potential Auger radiation therapy agent. Its incorporation in DNA of proliferating cells is enhanced by fluorodeoxyuridine. Here, we evaluated therapeutic activities of 125I-iododeoxyuridine in an optimized fluorodeoxyuridine pre-treatment inducing S-phase synchronization. Methods: After S-phase synchronization by fluorodeoxyuridine, cells were treated with 125I-iododeoxyuridine. Apoptosis analysis and S-phase synchronization were studied by flow cytometry. Cell survival was determined by colony-forming assay. Based on measured growth parameters, the number of decays per cell that induced killing was extrapolated. Results: Treatment experiments showed that 72 to 91% of synchronized cells were killed after 0.8 and 8 kBq/ml 125I-iododeoxyuridine incubation, respectively. In controls, only 8 to 38% of cells were killed by corresponding 125I-iododeoxyuridine activities alone and even increasing the activity to 80 kBq/ml gave only 42 % killing. Duplicated treatment cycles or repeated fluorodeoxyuridine pre-treatment allowed enhancing cell killing to >95 % at 8 kBq/ml 125I-iododeoxyuridine. About 50 and 160 decays per S-phase cells in controls and S-phase synchronization, respectively, were responsible for the observed cell killing at 0.8 kBq/ml radio-iododeoxyuridine. Conclusion: These data show the successful application of fluorodeoxyuridine that provided increased 125I-iododeoxyuridine Auger radiation cell killing efficacy through S-phase synchronization and high DNA incorporation of radio-iododeoxyuridine.