Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting.
Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1 and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-Tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.
oocyte; ubiquitin; proteasome; UCH
Although neurons are not productively infected with HIV-1, neuronal injury and death are frequently seen in the brains of AIDS patients with neurological and neurocognitive disorders. Evidently, viral proteins including Tat and cellular inflammatory factors released by activated and/or infected microglia, macrophages, and astrocytes contribute to neuronal cell death. Several studies have demonstrated that HIV-1 associated neuronal cell injury is mediated by dysregulation of signaling pathways that are controlled, in part, by a class of serine/threonine kinases. In this study we demonstrate that pDING, a novel plant-derived phosphate binding protein has the capacity to reduce the severity of injury and death caused by HIV-1 and its neurotoxic Tat protein. We demonstrate that pDING, also called p27SJ/p38SJ, protects cells from the loss of neuronal processes induced by Tat and promotes neuronal outgrowth after Tat-mediated injury. Further, expression of pDING prevents Tat-induced oxidative stress and mitochondrial permeability. With its profound phosphatase activity, pDING controls the activity of several kinases including MAPK, Cdk5 and their downstream target protein, MEF2, which is implicated in neuronal cell protection. Our results show that expression of pDING in neuronal cells diminishes the level of hyperphosphorylated forms of Cdk5 and MEF2 caused by Tat and the other neurotoxic agents that are secreted by the HIV-1 infected cells. These observations suggest that pDING, through its phosphatase activity, has the ability to manipulate the state of phosphorylation and activity of several factors involved in neuronal cell health in response to HIV-1.
HIV-1; DING; Neural degeneration
Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.
Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung.
Evolving data suggests that marrow hematopoietic stem cells show reversible changes in homing, engraftment and differentiation phenotype with cell cycle progression. Furthermore, marrow stem cells are a cycling population. Traditional concepts hold that the system is hierarchical, but the information on the lability of phenotype with cycle progression suggests a model in which stem cells are on a reversible continuum. Here we have investigated mRNA expression in murine lineage negative Stem Cell Antigen-1 positive stem cells of a variety of cell surface epitopes and transcription regulators associated with stem cell identity or regulation. At isolation these stem cells expressed almost all cell surface markers, and transcription factors studied, including receptors for G-CSF, GM-CSF and IL-7. When these stem cells were induced to transit cell cycle in vitro by exposure to interleukin-3 (IL-3), Il-6, IL-11 and steel factor some (CD34,CD45R c-kit, Gata-1, Gata-2, Ikaros and Fog) showed stable expression over time, despite previously documented alterations in phenotype, while others showed variation of expression between and within experiments. These included Sca-1, Mac-1, c-fms and c-mpl. Tal-1,endoglin and CD4 showed variation between experiments. These studies indicate that defined marrow stem cells express a wide variety of genes at isolation and with cytokine induced cell cycle transit show marked and reversible phenotype lability. Altogether, the phenotypic plasticity of gene expression for murine stem cells indicates a continuum model of stem cell regulation and extends the model to reversible expression with cell cycle transit of mRNA for cytokine receptors and stem cell markers.
Stem Cell; Phenotype; Hematopoiesis; Cell Cycle; Gene Expression; Plasticity
Drosophila can exhibit classic hallmarks of cancer, such as evasion of apoptosis, sustained proliferation, metastasis, prolonged survival, genome instability, and metabolic reprogramming, when cancer-related genes are perturbed. In the last two decades, studies in flies have identified several tumor suppressor and oncogenes. However, the greatest strength of the fly lies in its ability to model cancer hallmarks in a variety of tissue types, which enables the study of context-dependent tumorigenesis. We review the organs and tissues that have been used to model tumor formation, and propose new strategies to maximize the potential of Drosophila in cancer research.
Notch signaling is critical for skeletal muscle development and regeneration, permitting the expansion of progenitor cells by preventing premature differentiation. We have interrogated the pathways through which ligand-mediated signaling inhibits myogenesis by identifying Notch target genes and assessing their impact on differentiation in vitro. Notch activation led to the robust induction of the transcriptional repressors Hey1 and HeyL in myoblasts, but only constitutive expression of Hey1 blocked myogenesis. siRNA-mediated knockdown of Hey1 had no effect on Notch’s ability to inhibit differentiation, suggesting the existence of additional, possibly redundant pathways. We identified 82 genes whose expression was activated when C2C12 myoblasts were cultured in the presence of the Notch ligand Dll4. One of these, MyoR, is a novel Notch-responsive gene, whose protein product is known to repress myogenesis in vitro. siRNA-mediated knockdown of MyoR alone, or in combination with Hey1, was also ineffective at rescuing differentiation in the presence of Dll4. Our data support a model in which Notch signaling inhibits myogenesis through multiple pathways, two of which are defined by the Notch target genes Hey1 and MyoR.
Notch; Hey1; MyoR; MyoD; Myf-5; Myogenin; muscle; myogenesis
L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.
Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latter’s substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk-mediated signaling, but these effects do not correlate with LeTx-induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38-MK2-HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38-MK2-HSP27 in endothelial barrier permeability regulation.
Runx2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of Runx2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the Runx2+/− mice. In addition Runx2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the Runx2 protein in a phosphorylation-dependent manner and subsequently stabilizes Runx2 protein. In the absence of Pin1, Runx2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent Runx2 degradation. Collectively conformational change of Runx2 by Pin1 is essential for its protein stability and possibly enhances the level of active Runx2 in vivo.
Pin1; Runx2; cleidocranial dysplasia; peptidyl prolyl cis-trans isomerization; bone formation
Matrix metalloprotease-1 (MMP1) has been implicated in many human disease processes, however the lack of a well characterized murine homologue has significantly limited the study of MMP1 and the development of MMP-targeted therapeutics. The discovery of murine Mmp1a in 2001, the functional mouse homologue of MMP1, offers a valuable tool for modeling MMP1-mediated processes in mice. Variation in physiologic expression levels of Mmp1a in mice as compared to MMP1 in humans highlights the importance of understanding the similarities and differences between the homologues. Recent studies have demonstrated tumor growth-, invasion-, and angiogenesis-promoting functions of Mmp1a in lung cancer models, consistent with the analogous functions observed for human MMP1. Biochemical investigations have shown that point mutations in the pro-domain of mouse Mmp1a weaken docking between the pro- and catalytic domains, generating an unstable zymogen primed for activation. The difficulty to effectively maintain Mmp1a in the zymogen form may account for the tight control of Mmp1a expression and reduced expression in normal tissue as compared to inflammatory states or cancer. This discovery raises important questions about the activation mechanisms and regulation of the MMP family in general.
Polycyclic aromatic hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures of normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50–1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity.
Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.
Purified long-term multilineage repopulating marrow stem cells have been considered to be homogenous, but functionally these cells are heterogeneous. Many investigators urge clonal studies to define stem cells but, if stem cells are truly heterogeneous, clonal studies can only define heterogeneity. We have determined the colony growth and differentiation of individual lineage negative, rhodamine low, Hoechst low (LRH) stem cells at various times in cytokine culture, corresponding to specific cell cycle stages. These highly purified and cycle synchronized (98% in S phase at 40 h of culture) stem cells were exposed to two cytokine cocktails for 0, 18, 32, or 40 h and clonal differentiation assessed 14 days later. Total heterogeneity as to gross colony morphology and differentiation stage was demonstrated. This heterogeneity showed patterns of differentiation at different cycle times. These data hearken to previous suggestions that stem cells might be similar to radioactive isotopes; decay rate of a population of radioisotopes being highly predictable, while the decay of individual nuclei is heterogeneous and unpredictable (Till et al., 1964). Marrow stem cells may be most adequately defined on a population basis; stem cells existing in a continuum of reversible change rather than a hierarchy.
We recently reported a role of Polycomb repressive complex 2 (PRC2) and PRC2 trimethylation of histone 3 lysine 27 (H3K27me3) in the regulation of homeobox (HOX) (Marcinkiewicz and Gudas, 2013) gene transcript levels in human oral keratinocytes (OKF6-TERT1R) and tongue squamous cell carcinoma (SCC) cells. Here, we assessed both the levels of various histone modifications at a subset of homeobox genes and genome wide DNA methylation patterns in OKF6-TERT1R and SCC-9 cells by using ERRBS (enhanced reduced representation bisulfite sequencing). We detected the H3K9me3 mark at HOXB7, HOXC10, HOXC13 and HOXD8 at levels higher in OKF6-TERT1R than in SCC-9 cells; at IRX1 and SIX2 the H3K9me3 levels were conversely higher in SCC-9 than in OKF6-TERT1R. The H3K79me3 mark was detectable only at IRX1 in OKF6-TERT1R and at IRX4 in SCC-9 cells. The levels of H3K4me3 and H3K36me3 marks correlate with the transcript levels of the assessed homeobox genes in both OKF6-TERT1R and SCC-9. We detected generally lower CpG methylation levels on DNA in SCC-9 cells at annotated genomic regions which were differentially methylated between OKF6-TERT1R and SCC-9 cells; however, some genomic regions, including the HOX gene clusters, showed DNA methylation at higher levels in SCC-9 than OKF6-TERT1R. Thus, both altered histone modification patterns and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human oral cavity SCC cells, and this dysregulation potentially plays a role in the neoplastic phenotype of oral keratinocytes.
epigenetic silencing; oral squamous cell carcinoma; bisulfite-seq; homeobox; HOX clusters
The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL-2 family inhibitor) to enhance [sorafenib + HDAC inhibitor] toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of [sorafenib + HDAC inhibitor] exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K and mTOR activity and enhanced cleavage of pro-caspase 3 and reduced expression of MCL-1 and BCL-XL. Over-expression of BCL-XL or MCL-1 or expression of dominant negative pro-caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTORand to a lesser extent MEK1EE also protected cells that correlated with maintained c-FLIP-s expression, reduced BIM expression and increased BAD phosphorylation. In vivo [sorafenib + HDAC inhibitor] toxicity against tumors was increased in a greater than additive fashion by TRAIL.Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting [sorafenib + HDAC inhibitor] lethality.
sorafenib; histone deacetylase inhibitor; kinase; apoptosis; CD95; TRAIL; MCL-1
Core Binding Factor β (CBFβ) is complexed with the RUNX family of transcription factors in the nucleus to support activation or repression of genes related to bone (RUNX2), hematopoiesis (RUNX1) and gastrointestinal (RUNX3) development. Furthermore, RUNX proteins contribute to the onset and progression of different types of cancer. Although CBFβ localizes to cytoskeletal architecture, its biological role in the cytoplasmic compartment remains to be established. Additionally, the function and localization of CBFβ during the cell cycle are important questions relevant to its biological role. Here we show that CBFβ dynamically distributes in different stages of cell division and importantly is present during telophase at the midbody, a temporal structure important for successful cytokinesis. A functional role for CBFβ localization at the midbody is supported by striking defects in cytokinesis that include polyploidy and abscission failure following siRNA-mediated downregulation of endogenous CBFβ or overexpression of the inv(16) fusion protein CBFβ-SMMHC. Our results suggest that CBFβ retention in the midbody during cytokinesis reflects a novel function that contributes to epigenetic control.
Midbody; Core Binding Factor β; Cytokines
Viruses have been linked to approximately 20% of all human tumors worldwide. These transforming viruses encode viral oncoproteins that interact with cellular proteins to enhance viral replication. The transcriptional and post-transcriptional effects of these viral oncoproteins ultimately result in cellular transformation. Historically, viral research has been vital to the discovery of oncogenes and tumor suppressors with more current research aiding in unraveling some mechanisms of carcinogenesis. Interestingly, since transforming viruses affect some of the same pathways that are dysregulated in human cancers, their study enhances our understanding of the multistep process of tumorigenesis. This review will examine the cellular mechanisms targeted by oncogenic human viruses and the processes by which these effects contribute to transformation. In particular, we will focus on three transforming viruses, human T-cell leukemia virus type-I, hepatitis B virus and human papillomavirus. These viruses all encode specific oncogenes that promote cell cycle progression, inhibit DNA damage checkpoint responses and prevent programmed cell death in an effort to promote viral propagation. While the transforming properties of these viruses are probably unintended consequences of replication strategies, they provide excellent systems in which to study cancer development.
Adenosine is an endogenous metabolite that is released from all tissues and cells including liver, pancreas, muscle and fat, particularly under stress, intense exercise, or during cell damage. The role of adenosine in glucose homeostasis has been attributed to its ability to regulate, through its membrane receptors, processes such as insulin secretion, glucose release and clearance, glycogenolysis, and glycogenesis. Additionally, adenosine and its multiple receptors have been connected to lipid metabolism by augmenting insulin-mediated inhibition of lipolysis, and the subsequent increase in free fatty acids and glycerol levels. Furthermore, adenosine was reported to control liver cholesterol synthesis, consequently affecting plasma levels of cholesterol and triglycerides, and the amount of fat tissue. Alterations in the balance of glucose and lipid homeostasis have implications in both cardiovascular disease and diabetes. The ability of different adenosine receptors to activate and inhibit the same signaling cascades has made it challenging to study the influence of adenosine, adenosine analogs and their receptors in health and disease. This review focuses on the role and significance of different adenosine receptors in mediating the effect of adenosine on glucose and lipid homeostasis.
Adenosine; glucose; lipids
Proinflammatory cytokine TWEAK has now emerged as a key mediator of skeletal muscle-wasting in many catabolic conditions. However, the mechanisms by which TWEAK induces muscle proteolysis remain poorly understood. Here, we have investigated the role of ubiquitin-proteasome system, autophagy, and caspases in TWEAK-induced muscle wasting. Addition of TWEAK to C2C12 myotubes stimulated the ubiquitination of myosin heavy chain (MyHC) and augmented the expression of E3 ubiquitin ligase MuRF1. Pretreatment of myotubes with proteasome inhibitors MG132 or lactacystin or knockdown of MuRF1 by RNAi blocked the TWEAK-induced degradation of MyHC and myotube atrophy. TWEAK increased the expression of several autophagy-related molecules. Moreover, the inhibitors of autophagy improved the levels of MyHC in TWEAK-treated myotubes. TWEAK also increased activity of caspases in C2C12 myotubes. Pan-caspase or caspase 3 inhibitory peptide inhibited the TWEAK-induced loss of MyHC and myotube diameter. Our study demonstrates that nuclear factor-kappa B (NF-κB) transcription factor is essential for TWEAK-induced expression of MuRF1 and Beclin1. Furthermore, our results suggest that caspases contribute, at least in part, to the activation of NF-κB in response to TWEAK treatment. Collectively, the present study provides novel insight into the mechanisms of action of TWEAK in skeletal muscle.
Skeletal muscle atrophy; TWEAK; Ubiquitin-proteasome system; NF-kappa B; MuRF1; Capsases; Autophagy
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.
Lactoferricin; Interleukin-1; Lipopolysaccharide; Intervertebral disc degeneration; Anti-Inflammation; Anti-catabolic effect