The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-myc

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

Nutritional regulation of the fatty acid synthase promoter in vivo: Sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element

The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the −65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5′ regions are required for induction in vivo by fasting/refeeding and insulin; one at −278 to −131 albeit at a low level, and the other at −444 to −278 with an E-box at −332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5′ deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between −278 and −131 bp is required for SREBP function. We demonstrate that SREBP binds the −150 canonical SRE present between −278 and −131, and SREBP can function through the −150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the −278/−131 region and that the −150 SRE is the target sequence.

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-genii cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool. (c) 2006 Elsevier Inc. All rights reserved.

Polyunsaturated fatty acid requirements of murine colon adenocarcinomas

The polyunsaturated fatty acid (PUFA) requirements of three transplantable murine colon adenocarcinomas, the MAC13, MAC16 and MAC26, were evaluated in vitro and in vivo. When serum concentrations became growth limiting in vitro, proliferation of the MAC13 and MAC26 cell lines was stimulated by linoleic acid (LA) at 18μM and arachidonic acid (AA) at 16 or 33μM respectively. This was not demonstrated by the MAC16 cell line. MAC13 and MAC26 cells were found to be biochemically fatty acid deficient as measured by the formation of Mead acid (20:3 n-9), but the MAC16 cells were not. In vivo the growth of the MAC26 tumour was stimulated by daily oral administration of LA between 0.4-2.0g/kg. There was a threshold value of 0.4g/kg for the stimulation of MAC26 tumour growth, above which there was no further increase in tumour growth, and below which no increase in tumour growth was observed. This increased tumour growth was due to the stimulation of tumour cell proliferation in all areas of the tumour, with no effect on the cell loss factor. The growth of the MAC13, MAC16, and MAC26 cell lines in vitro were more effectively inhibited by lipoxygenase (LO) inhibitors than the cyclooxygenase inhibitor indomethacin. The specific 5-LO inhibitor Zileuton and the leukotriene D4 antagonist L-660,711 were less effective inhibitors of MAC cell growth in vitro than the less specific LO inhibitors BWA4C, BWB70C and CV6504. Studies of the hyroxyeicosatetraenoic acids (HETEs) produced from exogenous AA in these cells, suggested that a balance of eicosanoids produced from 5-LO, 12-LO and 15-LO pathways was required for cell proliferation. In vivo BWA4C, BWB70C and CV6504 demonstrated antitumour action against the MAC26 tumour between 20-50mg/kg/day. CV6504 also inhibited the growth of the MAC 13 tumour in vivo with an optimal effect between 5-10mg/kg/day. The antitumour action against the MAC16 tumour was also accompanied by a reduction in the tumour-induced host body weight loss at 10-25mg/kg/day. The antitumour action of CV6504 in all three tumour models was partially reversed by daily oral administration of 1.0g/kg LA. Studies of the AA metabolism in tumour homogenates suggested that this profound antitumour action, against what are generally chemoresistant tumours, was due to inhibition of eicosanoid production through LO pathways. As a result of these studies, CV6504 has been proposed for stage I./II. clinical trials against pancreatic cancer by the Cancer Research Campaign. This will be the first LO inhibitor entering the clinic as a therapeutic agent.

The central role of the neutrophil in ischaemic/reperfusion injury

Inadequate blood flow to an organ, ischaemia, may lead to both local and remote tissue injury characterized by oedema, increased microvascular permeability to protein and degradation of connective tissue components. This damage is probably caused by the accumulation and inappropriate activation of neutrophils which occurs when the tissue is reperfused. To test this hypothesis a number of in vitro models of the sequential stages of ischaemia/reperfusion injury were examined. Methods were initially developed to examine the adhesion of neutrophils to monolayers of a cultured endothelial cell line (ECV304) after periods of hypoxia and reoxygenation. Neutrophil migration in response to factors secreted by the treated endothelial cells was then assessed. The genesis of an inappropriate oxidative burst by the neutrophil upon exposure to endothelial chemoattractants and adhesion molecules was also measured. Finally to appraise how tissue function might be affected by endothelial cell hypoxia the contractility of vascular smooth muscle was examined. Neutrophil adhesion to ECV304 cells, which had been hypoxic for 4 hours and then reoxygenated for 30 minutes, was significantly increased. This response was probably initiated by reactive oxygen species (ROS) generated by the endothelial cells. Blockage of their production by allopurinol reduced the heightened adhesion. Similarly removal of ROS by superoxide dismutase or catalase also attenuated adhesion. ROS generation in turn caused the release of a soluble factor (s) which induced a conformational change on the neutrophil surface allowing it to bind to the intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell. Soluble factor (s) from hypoxia/reoxygenated endothelial cells also had a powerful neutrophil chemoattractant ability. When neutrophils were exposed to both hypoxic/reoxygenated endothelial cells and the soluble factor (s) released by them a large oxidative burst was elicited. This response was greatest immediately after reoxygenation and one hour later was diminishing suggesting at least one of the components involved was labile. Analysis of the supernatant from hypoxic/reoxygenated endothelial cell cultures and studies using inhibitors of secretion suggested platelet activating factor (PAF) may be a major component in this overall sequence of events. Lesser roles for IL-8, TNF and LTB4 were also suggested. The secretory products from hypoxia/reoxygenated endothelial cells also affected smooth muscle contractility having an anti-vasoconstrictor or relaxation property, similar to that exerted by PAF.

Factores preditivos de linfoma no síndrome de Sjögren : a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Eficacia de terapias biológicas de tipo anti-angiogénico en el cáncer colorrectal metastásico revisión sistemática de la literatura

Introducción: El cáncer colorrectal (CCR) se encuentra entre los 5 tipos de cáncer con mayor incidencia a nivel mundial. Alrededor del 20% de los casos son diagnosticados en estadios metastásico, donde el tratamiento inicialmente era quimioterapia con una supervivencia global a 5 años de 12 a 14 meses. Es así que se investiga el papel de la angiogénesis tumoral, orientado al desarrollo de terapias, implementando su uso en estadios avanzados. Metodología: Se realizó una búsqueda sistemática en las bases de datos Embase, PubMed, SciELO y LILIACS con términos estandarizados a través de la herramienta MeSH y DECS bajo los lineamientos establecidos en las guías de revisiones sistemáticas y meta-análisis (Manual Cochrane). Se tomaron estudios clínicos aleatorizados controlados con pacientes con CCR metastásico, que hayan recibido quimioterapia sola o combinada con terapias antiangiogénicas, publicados en inglés y español entre el 2003 y 2013. Resultados: 6 artículos cumplieron con criterios de inclusión. Estos reportaron 15.8 meses en promedio de supervivencia global en el tratamiento de quimioterapia asociada a terapias biológicas frente a 14.4 meses con solo quimioterapia. Los eventos adversos de tipo vascular aumentaron más en el grupo de antiangiogénicos, reportando muertes debidas a perforaciones intestinales. Conclusiones: Los regímenes de quimioterapia asociadas a terapias antiangiogénicas brindan una mayor supervivencia global y libre de progresión, al igual que mayor número de tasas de respuesta. Son terapias con eventos adversos importantes pero que deberá seleccionarse bien al paciente para disminuir su riesgo de eventos. Palabras claves: Cáncer colorrectal metastásico, terapia anti-angiogénica, quimioterapia en segunda línea, receptor del factor de crecimiento de endotelio vascular, supervivencia global.

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function.

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K(+) increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.

Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.

The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.

EPIDERMAL GROWTH-FACTOR AND TRANSFORMING GROWTH-FACTOR-ALPHA CHARACTERISTICS OF HUMAN ORAL-CARCINOMA CELL-LINES

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor a (TGF-a) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-a than normal cells. There was no statistical correlation between the autocrine production of TGF-a, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-cl were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-a to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta(1) in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.