Intracellular leucine-rich alpha-2-glycoprotein-1 competes with Apaf-1 for binding cytochrome c in protecting MCF-7 breast cancer cells from apoptosis.

Leucine-rich alpha-2-glycoprotein-1 (LRG1) has been shown to compete with apoptosis activating factor-1 (Apaf-1) for binding cytochrome c (Cyt c) and could play a role in inhibition of apoptosis. Employing MCF-7 breast cancer cells, we report that intracellular LRG1 does protect against apoptosis. Thus, cells transfected with the lrg1 gene and expressing higher levels of LRG1 were more resistant to hydrogen peroxide-induced apoptosis than parental cells, while cells in which LRG mRNA was knocked down by short hairpin (sh) RNA-induced degradation were more sensitive. The amount of Cyt c co-immunoprecipitated with Apaf-1 from the cytosol of apoptotic cells was inversely related to the level of LRG1 expression. In lrg1-transfected cells partially-glycosylated LRG1 was found in the cytosol and there was an increase in cytosolic Cyt c in live lrg1-transfected cells relative to parental cells. However, apoptosis was not spontaneously induced because Cyt c was bound to LRG1 and not to Apaf-1. Cyt c was the only detectable protein co-immunoprecipitated with LRG1. Following hydrogen peroxide treatment degradation of LRG1 allowed for induction of apoptosis. We propose that intracellular LRG1 raises the threshold of cytoplasmic Cyt c required to induce apoptosis and, thus, prevents onset of the intrinsic pathway in cells where Cyt c release from mitochondria does not result from committed apoptotic signaling. This mechanism of survival afforded by LRG1 is likely to be distinct from its extracellular survival function that has been reported by several research groups.

Extracellular renalase protects cells and organs by outside-in signalling.

Renalase was discovered as a protein synthesized by the kidney and secreted in blood where it circulates at a concentration of approximately 3-5 µg/ml. Initial reports suggested that it functioned as an NAD(P)H oxidase and could oxidize catecholamines. Administration of renalase lowers blood pressure and heart rate and also protects cells and organs against ischaemic and toxic injury. Although renalase's protective effect was initially ascribed to its oxidase properties, a paradigm shift in our understanding of the cellular actions of renalase is underway. We now understand that, independent of its enzymatic properties, renalase functions as a cytokine that provides protection to cells, tissues and organs by interacting with its receptor to activate protein kinase B, JAK/STAT, and the mitogen-activated protein kinase pathways. In addition, recent studies suggest that dysregulated renalase signalling may promote survival of several tumour cells due to its capacity to augment expression of growth-related genes. In this review, we focus on the cytoprotective actions of renalase and its capacity to sustain cancer cell growth and also the translational opportunities these findings represent for the development of novel therapeutic strategies for organ injury and cancer.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

IL-9, a local growth factor for synovial T cells in inflammatory arthritis.

OBJECTIVE: The regulatory role of the Th9 cells along with its signature cytokine IL-9 in human immune system and its aberrant activation in autoimmune diseases is currently under investigation. We are reporting the functional significance of IL-9 in the pathogenesis of autoimmune inflammatory arthritis. METHODS: CD3(+) T cells were obtained from peripheral blood (PB) and synovial fluid (SF) of psoriatic arthritis (PsA), rheumatoid arthritis (RA), and osteoarthritis (OA) patients. MTT, FACS based CFSE dilution assay and apoptosis assay (Annexin-V) were performed to determine the pro-growth/survival effect of human recombinant IL-9 on activated CD3(+) T cells. Immunoblots were performed to determine the signaling proteins responsible for the progrowth/survival effect of IL-9. RESULTS: SF of PsA and RA was enriched with IL-9 producing CD3(+) T cells compared to the SF in OA. IL-9 level measured by ELISA was significantly elevated in PsA and RA patients compared to SF in OA (<.001). Activated T cells of PsA and RA had higher levels of IL-9 receptors. IL-9 promoted proliferation and survival of the CD3(+) T cells of PB and SF of PsA and RA and compared to untreated (media) controls (p<.005, t-test). IL-9 induced proliferation of T cells was dependent on PI3K/Akt/mTOR signaling pathway. CONCLUSION: IL-9 is functionally active, and is a pro-growth/survival factor for the localized pathologic T cells in the synovium of inflammatory arthritis. The pro-growth/survival effect is mediated by the activation of mTOR kinase cascade. To our knowledge, this is the first report of a functional role of IL-9 in human autoimmune arthritis.

Deletion of afpab1 Causes Increased Sensitivity to Oxidative Stress and Hypovirulence in Aspergillus fumigatus.

Aspergillus fumigatus AFPAB1 is the ortholog of the Aspergillus oryzae cytoplasmic messenger ribonucleoprotein granules AOPAB1 that function to depress the initiation of translation during stress. A. fumigatus can regulate its cellular physiology in response to environmental stresses, but there has been no research on Pab1 in A. fumigatus. The associated gene afpab1 was replaced with a hygromycin-selectable marker to generate the strain Δafpab1. Phenotypic analysis showed that the Δafpab1 grew more weakly than the wild-type strain. Also the germination rate of Δafpab1 was decreased when exposed to oxidative stress. The morphology of Δafpab1 spores also showed great changes. The killing rate of Δafpab1 by RAW264.7 murine macrophage cells was increased, and the reactive oxygen species (ROS) scavenging ability of Δafpab1 was decreased. Pathogenicity testing showed that the deletion strain had decreased virulence. Therefore, we conclude that afpab1 activity is correlated with susceptibility to oxidative stress, and deletion of afpab1 from A. fumigatus possibly leads to observed hypovirulence in an immunosuppressed mouse model.

Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity.

Survival factor A (SvfA) in Aspergillus nidulans plays multiple roles in growth and developmental processes. It is a candidate for a novel VeA-dependent protein involved in sexual development. VeA is a key developmental regulator in Aspergillus species that can interact with other velvet-family proteins and enter into the nucleus to function as a transcription factor. In yeast and fungi, SvfA-homologous proteins are required for survival under oxidative and cold-stress conditions. To assess the role of SvfA in virulence in A. nidulans, cell wall components, biofilm formation, and protease activity were evaluated in a svfA-gene-deletion or an AfsvfA-overexpressing strain. The svfA-deletion strain showed decreased production of ß-1,3-glucan in conidia, a cell wall pathogen-associated molecular pattern, with a decrease in gene expression for chitin synthases and ß-1,3-glucan synthase. The ability to form biofilms and produce proteases was reduced in the svfA-deletion strain. We hypothesized that the svfA-deletion strain was less virulent than the wild-type strain; therefore, we performed in vitro phagocytosis assays using alveolar macrophages and analyzed in vivo survival using two vertebrate animal models. While phagocytosis was reduced in mouse alveolar macrophages challenged with conidia from the svfA-deletion strain, the killing rate showed a significant increase with increased extracellular signal-regulated kinase ERK activation. The svfA-deletion conidia infection reduced host mortality in both T-cell-deficient zebrafish and chronic granulomatous disease mouse models. Taken together, these results indicate that SvfA plays a significant role in the pathogenicity of A. nidulans.

[Messenger RNA for Cell Editing -Disease Treatment and Cell Transplantation Using Pro-survival mRNA].

Vigorous efforts are being made to manipulate cellular functions in a desirable manner for biomedical purposes. Recent advances in platform technologies have made cell editing achievable; this includes generation of induced pluripotent stem cells and chimeric antigen receptor T cells, as well as direct cell reprogramming. mRNA, as compared to DNA, is an excellent tool for potentiating cell editing technologies, owing to its distinct properties in gene introduction. Herein, hepatocytes were edited ex vivo and in vivo, by introducing pro-survival mRNA, to be resistant to cell death. DNA-based introduction of pro-survival gene poses safety concerns due to its genomic integration, as prolonged and uncontrolled expression of pro-survival proteins after the integration may promote cancer. In contrast, mRNA lacks such a risk. Moreover, mRNA-based introduction of Bcl-2, a pro-survival factor, was more effective in preventing the death of cultured hepatocytes than Bcl-2 plasmid DNA (pDNA) introduction. Mechanistically, mRNA induced protein expression in a larger percentage of the hepatocytes compared to pDNA, presumably because the process of pDNA nuclear entry in transfection is challenging. In hepatocyte transplantation to mouse liver, ex vivo introduction of Bcl-2 mRNA significantly improved the engraftment efficiency of the hepatocytes, leading to successful functional support of the liver in a mouse model of chronic hepatitis. Furthermore, in vivo administration of Bcl-2 mRNA exhibited an anti-apoptotic effect on the hepatocytes of a mouse model of fulminant hepatitis. These results demonstrate the potential advantages of mRNA introduction over DNA introduction in cell editing.

Survival factor 1 contributes to the oxidative stress response and is required for full virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen that infects over 400 species of plants worldwide. Reactive oxygen species (ROS) modulations are critical for the pathogenic development of S. sclerotiorum. The fungus applies enzymatic and non-enzymatic antioxidants to cope with the oxidative stress during the infection processes. Survival factor 1 was identified and characterized to promote survival under conditions of oxidative stress in Saccharomyes cerevisiae. In this research, a gene named SsSvf1 was predicted to encode a survival factor 1 homologue in S. sclerotiorum. SsSvf1 transcripts showed high expression levels in hyphae under oxidative stress. Silencing of SsSvf1 resulted in increased sensitivity to oxidative stress in culture and increased levels of intracellular ROS. Transcripts of SsSvf1 showed a dramatic increase during the initial stage of infection and the gene-silenced strains displayed reduced virulence on oilseed rape and Arabidopsis thaliana. Inhibition of plant ROS production partially restores virulence of SsSvf1 gene-silenced strains. SsSvf1 gene-silenced strains exhibited normal oxalate production, but were impaired in compound appressorium formation and cell wall integrity. The results suggest that SsSvf1 is involved in coping with ROS during fungal-host interactions and plays a crucial role in the pathogenicity of S. sclerotiorum.

Survival Factor Gene FgSvf1 Is Required for Normal Growth and Stress Resistance in Fusarium graminearum.

Survival factor 1 (Svf1) is a protein involved in cell survival pathways. In Saccharomyces cerevisiae, Svf1 is required for the diauxic growth shift and survival under stress conditions. In this study, we characterized the role of FgSvf1, the Svf1 homolog in the homothallic ascomycete fungus Fusarium graminearum. In the FgSvf1 deletion mutant, conidial germination was delayed, vegetative growth was reduced, and pathogenicity was completely abolished. Although the FgSvf1 deletion mutant produced perithecia, the normal maturation of ascospore was dismissed in deletion mutant. The FgSvf1 deletion mutant also showed reduced resistance to osmotic, fungicide, and cold stress and reduced sensitivity to oxidative stress when compared to the wild-type strain. In addition, we showed that FgSvf1 affects glycolysis, which results in the abnormal vegetative growth in the FgSvf1 deletion mutant. Further, intracellular reactive oxygen species (ROS) accumulated in the FgSvf1 deletion mutant, and this accumulated ROS might be related to the reduced sensitivity to oxidative stress and the reduced resistance to cold stress and fungicide stress. Overall, understanding the role of FgSvf1 in F. graminearum provides a new target to control F. graminearum infections in fields.

Prolonged engraftment of transplanted hepatocytes in the liver by transient pro-survival factor supplementation using ex vivo mRNA transfection.

Cell transplantation therapy needs engraftment efficiency improvement of transplanted cells to the host tissues. Ex vivo transfection of a pro-survival gene to transplanted cells is a possible solution; however prolonged expression and/or genomic integration of the gene can be cancer promoting. To supply pro-survival protein only when it is needed, we used mRNA transfection, which exhibits transient protein expression profiles without the risk of genomic integration. Ex vivo transfection of mRNA encoding Bcl-2, a pro-survival factor, led to enhanced hepatocyte engraftment in both of normal and diseased mouse liver, effectively supporting liver function in a model of chronic hepatitis. The transplanted hepatocytes maintained their viability and function in the liver for at least one month, though Bcl-2 expression from mRNA was sustained for just a few days. Mechanism analyses suggest that Bcl-2 inhibits Kupffer cell-mediated hepatocyte clearance, which occurs within 2 days after transplantation. Within 2 days, hepatocytes migrated to the liver parenchyma, presumably a suitable place for the hepatocytes to survive without Bcl-2 expression. Thus, the duration of Bcl-2 expression from mRNA was sufficient to achieve prolonged engraftment. Ex vivo mRNA transfection allows supply of pro-survival factors to transplanted cells with minimal safety concerns accompanying prolonged expression, providing an effective platform to improve engraftment efficiency in cell transplantation therapy.

Overexpression of CLEC3A promotes tumor progression and poor prognosis in breast invasive ductal cancer.

INTRODUCTION: The aim of this study was to evaluate the expression of C-type lectin domain family 3 member A (CLEC3A) and its clinical significance in breast invasive ductal cancer (IDC) as well as its effect on breast cancer (BC) cell proliferation and metastasis. In this study, the level of CLEC3A expression in The Cancer Genome Atlas (TCGA) datasets was analyzed. MATERIALS AND METHODS: Clinical collected samples and BC cells were measured using quantitative reverse transcription polymerase chain reaction. Its correlations with patients' clinicopathological characteristics were analyzed by Pearson's chi-squared test. Overall survival (OS) analysis was performed by the Kaplan-Meier method and Cox's proportional-hazards model. BC cell proliferation, migration, and invasion by CLEC3A knockdown were assessed using Cell Counting Kit-8 and colony formation assay, wound healing model and transwell assay, respectively, in BT474 cell line. Activities of survival factors and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling were measured by testing key molecules using Western blot assay. RESULTS: CLEC3A expression was markedly higher in breast IDC tissues than normal breast tissues or adjacent normal tissue. Patients with high CLEC3A expression related to higher lymph node and poorer OS of breast IDC. CLEC3A knockdown by siRNA could inhibit the BC cells BT474 proliferation, migration, and invasion, together with a decrease in expression of key proteins in survival factors and PI3K/AKT signaling pathway. CONCLUSION: Elevated CLEC3A expression may correlate with breast IDC metastatic potential and indicated a poor prognosis in breast IDC. CLEC3A knockdown inhibited BC cell growth and metastasis might be through suppressing PI3K/AKT signaling activity. These findings unravel that CLEC3A is a promising therapeutic target for BC in the future.

Identification of Genes Related to Fungicide Resistance in Fusarium fujikuroi.

We identified two genes related to fungicide resistance in Fusarium fujikuroi through random mutagenesis. Targeted gene deletions showed that survival factor 1 deletion resulted in higher sensitivity to fungicides, while deletion of the gene encoding F-box/WD-repeat protein increased resistance, suggesting that the genes affect fungicide resistance in different ways.

Presence of primary cilia in cancer cells correlates with prognosis of pancreatic ductal adenocarcinoma.

Primary cilia are microtubule-based organelles that protrude from basal bodies and are involved in cell differentiation, sensory functions, and planar cell polarity. Although there are many studies examining the roles of primary cilia in the fields of embryology and physiology, few such studies have been carried out in the field of oncology, and the role of primary cilia in cancer cells is poorly understood. In this study, we identified primary cilia by immunofluorescence analysis in which primary cilia were visualized as green rods labeled with anti-acetylated α-tubulin adjacent to basal bodies detected as red dots labeled with anti-γ-tubulin. Primary cilia were found in human pancreatic cancer cell lines and in cancer cells in 25 of 100 pancreatic ductal carcinoma patients. In the clinical samples, most primary cilia in cancer tissue were observed in areas showing well-differentiated glandular structures. Patients whose cancers were primary cilia positive had a higher frequency of lymph node metastasis than those whose cancers were primary cilia negative (P = .016). Univariate analysis demonstrated that tumor size (P = .009), tumor grade (P = .001), lymph node metastasis (P = .008), and the presence of primary cilia (P = .002) correlated with overall survival. Multivariate analysis found that tumor grade (P < .001) and the presence of primary cilia (P = .001) were independent prognostic indicators. In conclusion, we showed that pancreatic cancer cells can form primary cilia and that the presence of primary cilia is significantly associated with the prognosis of pancreatic ductal adenocarcinoma.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

Role of advanced glycation end products in cellular signaling.

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

The Effects of Insulin-like Growth Factor-I (IGF-I) in Mouse Lung Cancer Cells.

PURPOSE: Insulin-like growth factor-I (IGF-I) is an important mitogen in many types of malignancies. The purpose of this study was to evaluate the role of the IGF system on cell proliferation and cell death in mouse lung cancer cell lines (3LL). MATERIALS AND METHODS: Northern analysis was performed in 3LL cells. We evaluated the phosphorylation of IGF-I receptor (IGF-IR) with IGF-I stimulation. MTT assay was performed after treating 3LL cells with IGF-I and the treatment effect on cell death in the presence of anticancer drug was investigated. RESULTS: Northern analysis revealed the presence of IGF-I and IGF-IR mRNA expression in 3LL cells. IGF-I increased cellular proliferation in serum free media. IGF-I also stimulated the tyrosine phosphorylation of two proteins: one, with a molecular mass of 95 kDa, was the beta-subunit of IGF-IR; the other, with an approximate molecular mass of 185 kDa, was originally identified as the insulin receptor substrate-I (IRS-I). IGF-I at a low concentration inhibited the cell death induced by adriamycin. CONCLUSION: IGF-I, a mitogen through the phosphorylation of the IGF-IR beta-subunit, acts as a survival factor to inhibit cell death. Therefore, these findings suggest that IGF-I and IGF-IR are involved in both the cell proliferation and cell death associated with cancer cell growth.