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Articles from this Volume

Ke Pan, Xiao-ting Liang, Hua-kun Zhang, Jing-jing Zhao, Dan-dan Wang, Jian-jun Li, Qizhou Lian, Alfred E Chang, Qiao Li, and Jian-chuan Xia

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation
scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor. 

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Posted by Leah Caracappa on May 14, 2012 3:30 PM CDT
Adnana Paunel-Görgülü, Tamara Kirichevska, Tim Lögters, Joachim Windolf, and Sascha Flohé

Delayed neutrophil apoptosis and overshooting neutrophil activity contribute to organ dysfunction and subsequent organ failure in sepsis. Here, we investigated apoptotic signaling pathways that are involved in the inhibition of spontaneous apoptosis in neutrophils isolated from major trauma patients with uneventful outcome as well as in those with sepsis development. DNA fragmentation in peripheral blood neutrophils showed an inverse correlation with the organ dysfunction at d 10 after trauma in all patients, supporting the important role of neutrophil apoptosis regulation for patient's outcome. The expression of the antiapoptotic Bcl-2 protein members A1 and Mcl-1 were found to be diminished in the septic patients at d 5 and d 10 after trauma. This decrease was also linked to an impaired intrinsic apoptosis resistance, which has been previously shown to occur in neutrophils during systemic inflammation. In patients with sepsis development, delayed neutrophil apoptosis was found to be associated with a disturbed extrinsic pathway, as demonstrated by reduced caspase-8 activity and Bid truncation. Notably, the expression of Dad1 protein, which is involved in protein N-glycosylation, was significantly increased in septic patients at d 10 after trauma. Taken together, our data demonstrate that neutrophil apoptosis is regulated by both the intrinsic and extrinsic pathway, depending on patient's outcome. These findings might provide a molecular basis for new strategies targeting cell death pathways in apoptosisresistant neutrophils during systemic inflammation.

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Posted by Leah Caracappa on May 14, 2012 9:09 AM CDT
Christina Gross and Gary J Bassell

The fragile X mental retardation protein (FMRP) plays a key role for neurotransmitter-mediated signaling upstream of neuronal protein synthesis. Functional loss of FMRP causes the inherited intellectual disability fragile X syndrome (FXS), and leads to increased and stimulus-insensitive neuronal protein synthesis in FXS animal models. Previous studies suggested that excess protein synthesis mediated by dysregulated signal transduction contributes to the majority of neurological defects in FXS, and might be a promising target for therapeutic strategies in patients. However, possible impairments in receptor-dependent protein synthesis have not been evaluated in patient cells so far. Using quantitative fluorescent metabolic labeling, we demonstrate that protein synthesis is exaggerated and cannot be further increased by cytokine stimulation in human fragile X lymphoblastoid cells. Our previous work suggested that loss of FMRP-mediated regulation of protein expression and enzymatic function of the PI3K catalytic subunit p110β contributes to dysregulated protein synthesis in a mouse model of FXS. Here, we demonstrate that these molecular mechanisms are recapitulated in FXS patient cells. Furthermore, we show that treatment with a p110β-selective antagonist rescues excess protein synthesis in synaptoneurosomes from an FXS mouse model and in patient cells. Our work suggests that dysregulated protein synthesis and PI3K activity in patient cells might be suitable biomarkers to quantify the efficacy of drugs to ameliorate molecular mechanisms underlying FXS, and could be used for drug screens to refine treatment strategies for individual patients. Moreover, we provide rationale to pursue p110β-targeting treatments as potential therapy in FXS, and possibly other autism spectrum disorders.

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Posted by Leah Caracappa on May 14, 2012 9:07 AM CDT
Anja Weimer, Henning Madry, Jagadeesh K Venkatesan, Gertrud Schmitt, Janina Frisch, Anna Wezel,
Jochen Jung, Dieter Kohn, Ernest F Terwilliger, Stephen B Trippel, and Magali Cucchiarini

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)- mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while upregulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.

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Posted by Leah Caracappa on May 14, 2012 9:06 AM CDT
Kasey Davis, Sami Banerjee, Arnaud Friggeri, Celeste Bell, Edward Abraham, and Mourad Zerfaoui

Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.

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Posted by Leah Caracappa on May 14, 2012 9:05 AM CDT
Vaishali R Moulton, Dana R Holcomb, Melissa C Zajdel, and George C Tsokos

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex multifactorial pathogenesis. T lymphocytes play a critical role in disease pathogenesis and display abnormal gene expression and poor interleukin (IL)-2 production. We previously showed that the expression of the transcriptional repressor cyclic AMP response element modulator α (CREMα) is increased in SLE T cells and contributes to reduced IL-2 production. Although estrogen is implicated in the onset and exacerbation of SLE, the precise nature of molecular events regulated by estrogen in immune cell function is not well understood. Here, we asked whether estrogen regulates the expression of CREMα in human T lymphocytes. We show that exposure of human T cells to 17-β-estradiol leads to a dose-dependent increase in CREMα mRNA expression, and this increase appears to be mediated through the estrogen receptors α and β. We show that the increased expression of CREMα is due to increased transcriptional activity of the CREM promoter and is mediated by increased expression and binding of the Sp1 transcriptional activator. We further show that estrogen treatment leads to a dose-dependent decrease in IL-2 mRNA and cytokine production by T cells. Finally, the effect of β-estradiol on CREMα is observed more frequently in T cells from women than from men. We conclude that estrogen can modulate the expression of CREMα and lead to IL-2 suppression in human T lymphocytes, thus revealing a molecular link between hormones and the immune system in SLE.

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Posted by Leah Caracappa on May 14, 2012 9:03 AM CDT
Peedstroud9@gmail.comnelope Korkolopoulou, Georgia Levidou, Elias A El-Habr, Christos Adamopoulos, Vassilis Samaras, Athanasios Zisakis, Nikolaos Kavantzas, Efstathios Boviatsis, Paraskevi Fragkou, Athanasios G Papavassiliou, Efstratios Patsouris, and Christina Piperi

The aim was to expand recently published information regarding the significance of the interleukin (IL)-8/p-STAT-3 (signal transducer and activator of transcription) pathway in astrocytomas, focusing on the IL-8 receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), and the STAT-3 inhibitor SOCS-3 (suppressors of cytokine signaling). A total of 91 paraffin-embedded human astrocytoma tissues (grades II–IV) were investigated for the association of SOCS-3 and CXCR2 expression with clinicopathologic and morphometric microvascular characteristics, vascular endothelial growth factor (VEGF), IL-8 and p-STAT-3 expression and patient survival. Peripheral IL-8 secretion levels were assessed by enzyme-linked immunosorbent spot (ELISPOT). SOCS-3, p STAT-3 and CXCR2 protein levels were also quantified by Western immunoblotting in six cases, and the protein levels of SOCS-3 and CXCR2 were correlated with the immunohistochemical expression of the respective proteins. All CXCR2-positive cases by Western immunoblotting displayed increased peripheral IL-8 secretion levels. Treatment of primary glioblastoma cell cultures with exogenous IL-8 enhanced proliferation, and this effect was inhibited by treatment with a neutralizing anti-CXCR2 antibody. SOCS-3 and CXCR2 were expressed by neoplastic astrocytes in 92.4% and 48.78% of cases, respectively, with their levels increasing with histological grade and extent of necrosis. VEGF expression and microvessel density, CXCR2 and IL-8 levels were interrelated. SOCS-3 and p-STAT 3 were coexpressed in 85.7% of cases, although they were not interrelated. In univariate survival analysis, increased SOCS-3 expression and the presence of CXCR2 adversely affected survival, whereas in multivariate analysis, only CXCR2 remained significant. The prognostic significance of CXCR2 was validated in an independent set of 63 patients. Our data implicate IL-8/CXCR2 signaling pathway in the progression and regulation of angiogenesis in astrocytomas and provide a rationale for CXCR2 therapeutic exploitation in these tumors.

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Posted by Leah Caracappa on May 14, 2012 8:55 AM CDT
Liangjing Wang, Shujie Chen, Meng Xue, Jing Zhong, Xian Wang, Lihong Gan, Emily KY Lam,
Xin Liu, Jianbin Zhang, Tianhua Zhou, Jun Yu, Hongchuan Jin, and Jianmin Si

Homeobox D10 (HoxD10 ) gene plays a critical role in cell differentiation and morphogenesis during development. However, the function of HoxD10 in tumor progression remains largely unknown. We demonstrate that the expression of HoxD10 is commonly downregulated in gastric cancer tissues (n = 33) and cell lines (n = 8) relative to normal stomach tissues. Functionally, reexpression of HoxD10 results in significant inhibition of cell survival, induction of cell apoptosis, and impairment of cell migration and invasion. Moreover, ectopic expression of HoxD10 suppresses gastric tumor growth in a mouse xenograft model. To identify target candidates of HoxD10, we performed cDNA microarray and showed that HoxD10 regulates multiple downstream genes including IGFBP3. Reintroduction of HoxD10 transcriptionally upregulates IGFBP3, activates caspase 3 and caspase 8, and subsequently induces cell apoptosis. Methylation specific PCR revealed that HoxD10 promoter DNA was hypermethylated in gastric cancer cell lines. Additionally, 5-aza demethylation treatment could transiently reactivate the expression of HoxD10 in gastric cancer cells. HoxD10 promoter methylation frequently was detected in gastric cancer tissues obtained from endoscopic biopsies (85.7%, 24/28) and surgically resected samples (82.6%, 57/69). Intestinal metaplasia tissues showed a 60% methylation rate (18/30), but no detectable methylation in normal stomach tissues (0%, 0/10). Taken together, our results suggest that HoxD10 functions as a candidate tumor suppressor in gastric cancer, which is inactivated through promoter hypermethylation.


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Posted by Leah Caracappa on May 14, 2012 8:53 AM CDT
Laura Canzi, Valeria Castellaneta, Stefania Navone, Sara Nava, Marta Dossena, Ileana Zucca,
Tiziana Mennini, Paolo Bigini, and Eugenio A Parati

Mesenchymal stem cell (MSC) therapy is considered one of the most promising approaches for treating different neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). We previously characterized a subpopulation of human skeletal muscle–derived stem cells (SkmSCs) with MSC-like characteristics that differentiate into the neurogenic lineage in vitro. In the present study, we evaluated the SkmSC therapeutic effects in the most characterized model of spontaneous motor neuron degeneration, the Wobbler (Wr) mouse. Before evaluating the therapeutic efficacy in the Wr mouse, we followed the route of Skm- SCs at different times after intracerebroventricular injection. Two exogenous tracers, superparamagnetic iron oxide (SPIO) nanoparticles and Hoechst 33258, were used for the in vivo and ex vivo tracking of SkmSCs. We found that the loading of both Hoechst and SPIO was not toxic and efficiently labeled SkmSCs. The magnetic resonance imaging (MRI) system 7 Tesla allowed us to localize transplanted SkmSCs along the whole ventricular system up to 18 wks after injection. The ex vivo Hoechst 33258 visualization confirmed the in vivo results obtained by MRI analyses. Behavioral observations revealed a fast and sustained improvement of motor efficacy in SkmSC-treated Wr mice associated with a relevant protection of functional neuromuscular junctions. Moreover, we found that in SkmSC-treated Wr mice, a significant increase of important human antiinflammatory cytokines occurred. This evidence is in accordance with previous findings showing the bystander effect of stem cell transplantation in neurodegenerative disorders and further strengthens the hypothesis of the possible link between inflammation, cytotoxicity and ALS.

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Posted by Leah Caracappa on May 14, 2012 8:47 AM CDT
Yi-Jen Liao, Tzu-Lang Chen, Tzong-Shyuan Lee, Hsiang-An Wang, Chung-Kwe Wang, Li-Ying Liao,
Ren-Shyan Liu, Shiu-Feng Huang, and Yi-Ming Arthur Chen

Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt–/–) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt–/– mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with 131I-labeled 6β-iodocholesterol demonstrated that Gnmt–/– mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt–/– mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81–105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171–295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.

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Posted by Leah Caracappa on May 14, 2012 8:45 AM CDT
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