Effect of a Blueberry-Derived Antioxidant Matrix on Infrared-A Induced Gene Expression in Human Dermal Fibroblasts.

There is compelling evidence that Infrared A (IRA) from natural sunlight contributes to photoaging of human skin by inducing the expression of matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Corresponding mechanistic studies have shown that IRA does so by increasing the production of reactive oxygen species in irradiated cells. In the present study, we therefore asked if treatment of primary human skin fibroblasts with a blueberry-derived antioxidant matrix (BerrimatrixTM), which is employed as an active ingredient in commercially available skin care products that are topically applied, can prevent IRA-induced MMP-1 expression in these cells. In this in vitro study, we have found that this antioxidant containing matrix is well tolerated by fibroblast over a broad concentration range and that it efficiently prevents IRA-induced MMP-1 mRNA expression. It may thus be speculated that topical application of this antioxidant containing matrix may be efficient in protecting human skin against IRA-induced wrinkle formation.

J Drugs Dermatol. 2017;16(8 Suppl 2):s125-128.

Integrative functional genomics of hepatitis C virus infection identifies host dependencies in complete viral replication cycle.

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

AKT2 confers protection against aortic aneurysms and dissections.

RATIONALE: Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown. OBJECTIVE: In this study, we examined the role of AKT2 in protecting the aorta from developing AAD. METHODS AND RESULTS: AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II, these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. In addition, angiotensin II-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase-9 (MMP-9) and reduced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1). In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 to the MMP-9 and TIMP-1 promoters. CONCLUSIONS: Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.

SHP-1 as a critical regulator of Mycoplasma pneumoniae-induced inflammation in human asthmatic airway epithelial cells.

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase ß-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

Calcifying human aortic smooth muscle cells express different bone alkaline phosphatase isoforms, including the novel B1x isoform.

BACKGROUND: Vascular calcification, causing cardiovascular morbidity and mortality, is associated with hyperphosphatemia in chronic kidney disease (CKD). In vitro, phosphate induces transdifferentiation of vascular smooth muscle cells to osteoblast-like cells that express alkaline phosphatase (ALP). In vivo, raised serum ALP activities are associated with increased mortality. A new bone ALP isoform (B1x) has been identified in serum from CKD patients. The present study investigated the different ALP isoforms in calcifying human aortic smooth muscle cells (HAoSMCs). METHODS: HAoSMCs were cultured for 30 days in medium containing 5 or 10 mmol/l ß-glycerophosphate in the presence or absence of the ALP-specific inhibitor tetramisole. RESULTS: All known bone-specific ALP (BALP) isoforms (B/I, B1x, B1 and B2) were identified in HAoSMCs. ß-Glycerophosphate stimulated calcification of HAoSMCs, which was associated with increased BALP isoforms B/I, B1x and B2. Tetramisole inhibited the ß-glycerophosphate-induced HAoSMC calcification, which was paralleled by the inhibition of the B1x and B/I, but not the other isoforms. CONCLUSIONS: HAoSMCs express the four known BALP isoforms. B/I, B1x and B2 could be essential for soft tissue calcification. B/I and B1x were more affected by tetramisole than the other isoforms, which suggests different biological functions during calcification of HAoSMCs.

Differential roles of JNK, ERK1/2, and p38 mitogen-activated protein kinases on endothelial cell tissue repair functions in response to tumor necrosis factor-α.

Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µM) or ERK1/2 (PD98059, 5 µM) significantly inhibited TNF-α-stimulated cell survival, contraction of collagen gels, and wound closure. In contrast, the p38 inhibitor SB203580 (5 µM) further amplified all of the TNF-α effects on HPAECs. TNF-α specifically activated p38α but not other p38 isoforms and suppression of p38α by an siRNA resulted in further amplification of the TNF-α effect. These results suggest that TNF-α stimulates tissue repair functions of HPAECs, and this may be mediated, at least in part, positively via JNK and ERK1/2, and negatively through p38α. MAPKs may play a role in endothelial cell-mediated tissue repair, especially in an inflammatory milieu where TNF-α is present.

Antidepressants inhibit DNA methyltransferase 1 through reducing G9a levels.

The discovery of epigenetic processes as possible pivotal regulatory mechanisms in psychiatric diseases raised the question of how psychoactive drugs may impact the epigenetic machinery. In the present study we set out to explore the specificity and the mode of action of the reported inhibitory effect of the TCA (tricyclic antidepressant) amitriptyline on DNMT (DNA methyltransferase) activity in primary astrocytes from the rat cortex. We found that the impact on DNMT was shared by another TCA, imipramine, and by paroxetine, but not by venlafaxine or the mood stabilizers carbamazepine and valproic acid. DNMT activity in subventricular neural stem cells was refractory to the action of ADs (antidepressants). Among the established DNMTs, ADs primarily targeted DNMT1. The reduction of enzymatic DNMT1 activity was neither due to reduced DNMT1 expression nor due to direct drug interference. We tested putative DNMT1-inhibitory mechanisms and discovered that a known stimulator of DNMT1, the histone methyltransferase G9a, exhibited decreased protein levels and interactions with DNMT1 upon AD exposure. Adding recombinant G9a completely reversed the AD repressive effect on DNMT1 function. In conclusion, the present study presents a model where distinct ADs affect DNMT1 activity via G9a with important repercussions for possible novel treatment regimes.

NADPH oxidase 4 promotes endothelial angiogenesis through endothelial nitric oxide synthase activation.

UNLABELLED: BACKGROUND- Reactive oxygen species serve signaling functions in the vasculature, and hypoxia has been associated with increased reactive oxygen species production. NADPH oxidase 4 (Nox4) is a reactive oxygen species-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia. METHODS AND RESULTS: Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the vascular endothelial cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hindlimb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by reactive oxygen species, we probed the effect of Nox4 on eNOS. In cultured endothelial cells overexpressing Nox4, we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4, we crossed our transgenic Nox4 mice with eNOS(-/-) mice. Aortas from these mice did not demonstrate enhanced aortic sprouting, and VECad-Nox4 mice on the eNOS(-/-) background did not demonstrate enhanced recovery from hindlimb ischemia. CONCLUSIONS: Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.

Immune complexes and late complement proteins trigger activation of Syk tyrosine kinase in human CD4(+) T cells.

In systemic lupus erythematosus (SLE), the autoantibodies that form immune complexes (ICs) trigger activation of the complement system. This results in the formation of membrane attack complex (MAC) on cell membrane and the soluble terminal complement complex (TCC). Hyperactive T cell responses are hallmark of SLE pathogenesis. How complement activation influences the T cell responses in SLE is not fully understood. We observed that aggregated human γ-globulin (AHG) bound to a subset of CD4(+) T cells in peripheral blood mononuclear cells and this population increased in the SLE patients. Human naive CD4(+) T cells, when treated with purified ICs and TCC, triggered recruitment of the FcRγ chain with the membrane receptor and co-localized with phosphorylated Syk. These events were also associated with aggregation of membrane rafts. Thus, results presented suggest a role for ICs and complement in the activation of Syk in CD4(+) T cells. Thus, we propose that the shift in signalling from ζ-chain-ZAP70 to FcRγ chain-Syk observed in T cells of SLE patients is triggered by ICs and complement. These results demonstrate a link among ICs, complement activation and phosphorylation of Syk in CD4(+) T cells.

Critical role of Src-Syk-PLC{gamma}2 signaling in megakaryocyte migration and thrombopoiesis.

Migration of megakaryocytes (MKs) from the proliferative osteoblastic niche to the capillary-rich vascular niche is essential for proplatelet formation and platelet release. In this study, we explore the role of surface glycoprotein receptors and signaling proteins in regulating MK migration and platelet recovery after immune-induced thrombocytopenia. We show that spreading and migration of mouse primary bone marrow-derived MKs on a fibronectin matrix are abolished by the Src family kinases inhibitor PP1, the Syk kinase inhibitor R406 and the integrin alphaIIbbeta3 antagonist lotrafiban. We also demonstrate that these responses are inhibited in primary phospholipase C gamma2 (PLCgamma2)-deficient MKs. Conversely, MK spreading and migration were unaltered in the absence of the collagen receptor, the glycoprotein VI-FcRgamma-chain complex. We previously reported a correlation between a defect in MK migration and platelet recovery in the absence of platelet endothelial cell adhesion molecule-1 and the tyrosine phosphatase CD148. This correlation also holds for mice deficient in PLCgamma2. This study identifies a model in which integrin signaling via Src family kinases and Syk kinase to PLCgamma2 is required for MK spreading, migration, and platelet formation.

1alpha,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells.

The current study presents data indicating that 1alpha,25-dihydroxyvitamin D(3) affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1alpha,25-dihydroxyvitamin D(3) suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3beta-hydroxysteroid dehydrogenase (3betaHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1alpha,25-dihydroxyvitamin D(3). Interestingly, the two CYP17A1-mediated activities were influenced reciprocally - the 17alpha-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1alpha,25-dihydroxyvitamin D(3)-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1alpha,25-dihydroxyvitamin D(3)-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

CXCL4-induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1.

Human monocytes respond to a variety of stimuli with a complex spectrum of activities ranging from acute defense mechanisms to cell differentiation or cytokine release. However, the individual intracellular signaling pathways related to these functions are not well understood. CXC chemokine ligand 4 (CXCL4) represents a broad activator of monocytes, which induces acute as well as delayed activities in these cells including cell differentiation, survival, or the release of ROS, and cytokines. Here, we report for the first time that CXCL4-treated monocytes significantly upregulate sphingosine kinase 1 (SphK1) mRNA and that CXCL4 induces SphK1 enzyme activity as well as its translocation to the cell membrane. Furthermore, we could show that pharmacological inhibition of SphK results in reversal of CXCL4-induced monocyte survival, cytokine expression, and release of oxygen radicals, which was confirmed by the use of SphK1-specific siRNA. CXCL4-mediated rescue from apoptosis, which is accompanied by inhibition of caspases, is controlled by SphK1 and its downstream element Erk. Taken together, these data assign SphK1 as a central regulator of acute and delayed monocyte activation and suggest SphK1 as a potential therapeutic target to suppress pro-inflammatory responses induced by CXCL4.

The cyclin-dependent kinase inhibitor R-roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis.

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non-inflammatory phagocytosis by professional phagocytes. Recently, cyclin-dependent kinase (CDK) inhibitors (e.g. R-roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF-kappaB and MAPK pathways and key endogenous survival proteins (typified by Mcl-1) are involved in the regulation of neutrophil apoptosis and, in cancer-cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R-roscovitine over-rides TNF-alpha and LPS-induced survival (determined by morphological examination and binding of fluorescently labelled annexin-V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF-kappaB activation (assessed by cytoplasmic IkappaBalpha proteolysis and NF-kappaB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down-regulation (at protein and mRNA level) of the survival protein Mcl-1 but not the pro-apoptotic bcl-2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Distinct roles of stress-activated protein kinases in Fanconi anemia-type C-deficient hematopoiesis.

The underlying molecular mechanisms that promote bone marrow failure in Fanconi anemia are incompletely understood. Evidence suggests that enhanced apoptosis of hematopoietic precursors is a major contributing factor. Previously, enhanced apoptosis of Fanconi anemia type C-deficient (Fancc(-/-)) progenitors was shown to involve aberrant p38 MAPK activation. Given the importance of c-Jun N-terminal kinase (JNK) in the stress response, we tested whether enhanced apoptosis of Fancc(-/-) cells also involved altered JNK activation. In Fancc(-/-) murine embryonic fibroblasts, tumor necrosis factor alpha (TNF-alpha) induced elevated JNK activity. In addition, JNK inhibition protected Fancc(-/-) murine embryonic fibroblasts and c-kit(+) bone marrow cells from TNF-alpha-induced apoptosis. Importantly, hematopoietic progenitor assays demonstrated that JNK inhibition enhanced Fancc(-/-) colony formation in the presence of TNF-alpha. Competitive repopulation assays showed that Fancc(-/-) donor cells cultured with the JNK inhibitor had equivalent levels of donor chimerism compared with Fancc(-/-) donor cells cultured with vehicle control. In contrast, culturing Fancc(-/-) cells with a p38 MAPK inhibitor significantly increased repopulating ability, supporting an integral role of p38 MAPK in maintaining Fancc(-/-) hematopoietic stem cell function. Taken together, these data suggest that p38 MAPK, but not JNK, has a critical role in maintaining the engraftment of Fancc(-/-)-reconstituting cells under conditions of stress.

Stem cell based cancer gene therapy.

The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Suicide gene therapy using genetically engineered mesenchymal stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. This review provides an explanation of the stem cell-targeted prodrug cancer gene therapy principle, with focus on the choice of prodrug, properties of bone marrow and adipose tissue-derived mesenchymal stem and neural stem cells as well as the mechanisms of their tumor homing ability. Therapeutic achievements of the cytosine deaminase/5-fluorocytosine prodrug system and Herpes simplex virus thymidine kinase/ganciclovir are discussed. In addition, delivery of immunostimulatory cytokines, apoptosis inducing genes, nanoparticles and antiangiogenic proteins by stem cells to tumors and metastases is discussed as a promising approach for antitumor therapy. Combinations of traditional, targeted and stem cell-directed gene therapy could significantly advance the treatment of cancer.

Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury.

Metabolic signaling through the posttranslational linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins represents a unique signaling paradigm operative during lethal cellular stress and a pathway that we and others have recently shown to exert cytoprotective effects in vitro and in vivo. Accordingly, the present work addresses the contribution of the hexosaminidase responsible for removing O-GlcNAc (ie, O-GlcNAcase) from proteins. We used pharmacological inhibition, viral overexpression, and RNA interference of O-GlcNAcase in isolated cardiac myocytes to establish its role during acute hypoxia/reoxygenation. Elevated O-GlcNAcase expression significantly reduced O-GlcNAc levels and augmented posthypoxic cell death. Conversely, short interfering RNA directed against, or pharmacological inhibition of, O-GlcNAcase significantly augmented O-GlcNAc levels and reduced posthypoxic cell death. On the mechanistic front, we evaluated posthypoxic mitochondrial membrane potential and found that repression of O-GlcNAcase activity improves, whereas augmentation impairs, mitochondrial membrane potential recovery. Similar beneficial effects on posthypoxic calcium overload were also evident. Such changes were evident without significant alteration in expression of the major putative components of the mitochondrial permeability transition pore (ie, voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D). The present results provide definitive evidence that O-GlcNAcase antagonizes posthypoxic cardiac myocyte survival. Moreover, such results support a renewed approach to the contribution of metabolism and metabolic signaling to the determination of cell fate.

Caspase 3 blocking avoids the expression of autoantigens triggered by apoptosis in neonatal Balb/c mice skin.

OBJECTIVE: To assess the effect of caspase 3 inhibition, in the expression of intracellular antigens induced by apoptosis. MATERIAL AND METHODS: Skin explants of neonatal Balb/c mice were used to assess the autoantigen expression. Skin was obtained by punch biopsies, tissues were cultured in DMEM; cell death was induced by chemicals and assessed by TUNEL. The expression of La, Ro, Sm, RNP, Cajal Bodies and NuMa antigens were monitored by immunohistochemistry using autoantibodies or monoclonal antibodies against these antigens. RESULTS: Chemicals used to induce cell death, successfully produced apoptosis or necrosis in more than 60% of keratinocytes, and viability was significantly decreased when it was compared with those in controls. An increased expression of all skin intracellular antigens in skin biopsies treated with chemicals, major antigenic expression was detected with anti-La and anti-Ro antibodies. The caspase 3 inhibitor DEVD-CMK significantly decreased the expression of antigens induced by chemicals. CONCLUSION: By this result we can infer that caspase inhibitors modify apoptosis and decrease the autoantigens associated to cell death.