Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

(+)alpha-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

The vitamin E derivative (+)alpha-tocopheryl succinate (alpha-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RAR alpha transgenic mice, we demonstrated that alpha-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that alpha-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, alpha-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for alpha-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy. Leukemia (2012) 26, 451-460; doi:10.1038/leu.2011.216; published online 26 August 2011

Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and Its Modulation by Ischemic Pre-Conditioning or Hemin Treatment

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

Mechanism and Efficiency of Cell Death of Type II Photosensitizers: Effect of Zinc Chelation

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment.

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Calegari VC, Abrantes JL, Silveira LR, Paula FM, Costa JM Jr, Rafacho A, Velloso LA, Carneiro EM, Bosqueiro JR, Boschero AC, Zoppi CC. Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets. J Appl Physiol 112: 711-718, 2012. First published December 15, 2011; doi:10.1152/japplphysiol.00318.2011.-Endurance training has been shown to increase pancreatic beta-cell function and mass. However, whether exercise modulates beta-cell growth and survival pathways signaling is not completely understood. This study investigated the effects of exercise on growth and apoptotic markers levels in rat pancreatic islets. Male Wistar rats were randomly assigned to 8-wk endurance training or to a sedentary control group. After that, pancreatic islets were isolated; gene expression and the total content and phosphorylation of several proteins related to growth and apoptotic pathways as well as the main antioxidant enzymes were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Reactive oxygen species (ROS) production was measured by fluorescence. Endurance training increased the time to reach fatigue by 50%. Endurance training resulted in increased protein phosphorylation content of AKT (75%), AKT substrate (AS160; 100%), mTOR (60%), p70s6k (90%), and ERK1/2 (50%), compared with islets from control group. Catalase protein content was 50% higher, whereas ROS production was 49 and 77% lower in islets from trained rats under basal and stimulating glucose conditions, respectively. Bcl-2 mRNA and protein levels increased by 46 and 100%, respectively. Bax and cleaved caspase-3 protein contents were reduced by 25 and 50% in islets from trained rats, respectively. In conclusion, these results demonstrate that endurance training favors the beta-cell growth and survival by activating AKT and ERK1/2 pathways, enhancing antioxidant capacity, and reducing ROS production and apoptotic proteins content.

Anticancer Effects of Synthetic Phosphoethanolamine on Ehrlich Ascites Tumor: An Experimental Study

Background: Antineoplastic phospholipids (ALPs) represent a promising class of drugs with a novel mode of action undergoes rapid turnover in the cell membrane of tumors, interfering with lipid signal transduction, inducing cell death. The aim of this study was to investigate the synthetic phosphoethanolamine (Pho-s) as a new anticancer agent. Materials and Methods: Cell viability and morphology were assessed by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst and rhodamine staining. Apoptosis was assessed by Annexin V and propidium iodide (PI) staining, caspase-3 activity, mitochondrial membrane potential (Delta m psi) and cell cycle analysis, combined with evaluation of tumor growth in Ehrlich Ascites Tumor (EAT) bearing mice. Results: We found that Pho-s 2.30 mg/ml induced cytotoxicity in all tumor cell lines studied without affecting normal cells. In vitro studies with EAT cells indicated that Pho-s induced apoptosis, demonstrated by an increase in Annexin-V positive cells, loss of mitochondrial potential (Delta m psi) and increased caspase-3 activity. It was also shown to increase the sub-G(1) apoptotic fraction and inhibit progression to the S phase of the cell cycle. Additionally, antitumor effects on the EAT-bearing mice showed that Pho-s, at a concentration of 35 and 70 mg/kg, inhibited tumor growth and increased the lifespan of animals without causing liver toxicity. Conclusion: These findings suggest that Pho-s is a potential anticancer candidate drug.

Effect of Cholecalciferol as Adjunctive Therapy With Insulin on Protective Immunologic Profile and Decline of Residual beta-Cell Function in New-Onset Type 1 Diabetes Mellitus

Objective: To evaluate the effect of vitamin D-3 on cytokine levels, regulatory T cells, and residual beta-cell function decline when cholecalciferol (vitamin D-3 administered therapeutically) is given as adjunctive therapy with insulin in new-onset type 1 diabetes mellitus (T1DM). Design and Setting: An 18-month (March 10, 2006, to October 28, 2010) randomized, double-blind, placebo-controlled trial was conducted at the Diabetes Center of Sao Paulo Federal University, Sao Paulo, Brazil. Participants: Thirty-eight patients with new-onset T1DM with fasting serum C-peptide levels greater than or equal to 0.6 ng/mL were randomly assigned to receive daily oral therapy of cholecalciferol, 2000 IU, or placebo. Main Outcome Measure: Levels of proinflammatory and anti-inflammatory cytokines, chemokines, regulatory T cells, hemoglobin A(1c), and C-peptide; body mass index; and insulin daily dose. Results: Mean (SD) chemokine ligand 2 (monocyte chemoattractant protein 1) levels were significantly higher (184.6 [101.1] vs 121.4 [55.8] pg/mL) at 12 months, as well as the increase in regulatory T-cell percentage (4.55%[1.5%] vs 3.34%[1.8%]) with cholecalciferol vs placebo. The cumulative incidence of progression to undetectable (<= 0.1 ng/mL) fasting C-peptide reached 18.7% in the cholecalciferol group and 62.5% in the placebo group; stimulated C-peptide reached 6.2% in the cholecalciferol group and 37.5% in the placebo group at 18 months. Body mass index, hemoglobin A(1c) level, and insulin requirements were similar between the 2 groups. Conclusions: Cholecalciferol used as adjunctive therapy with insulin is safe and associated with a protective immunologic effect and slow decline of residual beta-cell function in patients with new-onset T1DM. Cholecalciferol may be an interesting adjuvant in T1DM prevention trials.

In vivo study of laser irradiation of fractionated drug administration based mechanism for effective photodynamic therapy in rat liver

Up-regulation of stress-activated proteins in cancer cells plays a protective role against photodynamic induced apoptosis. Post photodynamic therapy extracted normal rat liver tissue usually shows a fraction of surviving cells, the photodynamic resistant cells, residing in the necrotic region. To treat these photo-dynamic resistant cells a technique has been proposed based on fractionated drug administration of diluted photosensitizer, keeping the net concentration (5 mg/kg) constant, and subsequently varying drug light interval (DLI). Flourescence measurements were made for the presence of photosensitizer in a tissue. For qualitative analysis both histological and morphological studies were made. Although preliminary aim of this approach was not achieved but there were some interesting observation made i.e. for higher dilution of photosensitizer there was a sharp boundary between necrotic and normal portion of tissue. An increase in the absorption coefficient (alpha) from 2.7 -> 2.9 was observed as photosensitizer was diluted while the corresponding threshold dose (D (th)) persistently decreases from (0.10 -> 0.02) J/cm(2) when irradiated with a 635 nm laser fluence of 150 J/cm(2).

Activation of Survival and Apoptotic Signaling Pathways in Lymphocytes Exposed to Palmitic Acid

The toxicity of palmitic acid (PA) towards a human T-lymphocyte cell line (Jurkat) has been previously investigated but the mechanism(s) of PA action were unknown. In the current study, Jurkat cells were treated with sub-lethal concentrations of PA (50-150 mu M) and the activity of various signaling proteins was investigated. PA-induced apoptosis and mitochondrial dysfunction in a dose-dependent manner as evaluated by DNA fragmentation assay and depolarization of the mitochondrial membrane, respectively. PA treatment provoked release of cytochrome c from the inner mitochondrial membrane to the cytosol, activated members of the MAPK protein family JNK, p38, ERK, activated caspases 3/9, and increased oxidative/nitrosative stress. Exposure of cells to PA for 12 h increased insulin receptor (IR) and GLUT-4 levels in the plasma membrane. Insulin treatment (10 mU/ml/30 min) increased the phosphorylation of the IR beta-subunit and Akt. A correlation was found between DNA fragmentation and expression levels of both IR and GLUT-4. Similar results were obtained for PA-treated lymphocytes from healthy human donors and from mesenteric lymph nodes of 48-h starved rats. PA stimulated glucose uptake by Jurkat cells (in the absence of insulin), stimulated accumulation of neutral lipids (triglyceride), and other lipid classes (phospholipids and cholesterol ester) but reduced glucose oxidation. Our results suggest that parameters of insulin signaling and non-oxidative glucose metabolism are stimulated as part of a coordinated response to prompt survival in lymphocytes exposed to PA but at higher concentrations, apoptosis prevails. These findings may explain aspects of lymphocyte dysfunction associated with diabetes. J. Cell. Physiol. 227: 339-350, 2012. (C) 2011 Wiley Periodicals, Inc.

Evidenze molecolari del ruolo antiproliferativo e pro-apoptotico della clusterina in cellule epiteliali prostatiche umane

The Clusterin (CLU) gene produces different forms of protein products which vary in their biological properties and distribution within the cell. Both the extra- and intracellular CLU forms regulate cell proliferation and apoptosis. Dis-regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over-expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC-3 prostate cancer cells. Following siRNA, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e. H3 mRNA, PCNA and cyclins A, B1 and D) as detected by RT-qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin–proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half-life is less than 2 hours. All CLU protein products were found poly-ubiquitinated by co-immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, strongly inducing the nuclear form of CLU (nCLU) and committing cells to caspase-dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumour suppressor factor.

Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)]. ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS and ARMS, but the MYCN amplification and expression levels shows a significant correlation and are greater in ARMS, in which they are associated with adverse outcome. We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28 and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated- PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced or repressed, with both genes previously described as targets of N-myc or Myc, and new genes undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility, metastasis, angiogenesis and muscle development). The changes in the expression of the most relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity), whereas treatment with the mutated-PNA had no effect. Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS, particularly for poor prognosis ARMS, and of other MYCN-expressing tumors.

Zinco, invecchiamento e sistema immunitario: effetti sull'apoptosi e sulla proliferazione dei linfociti

Immunosenescence is characterized by a complex remodelling of the immune system, mainly driven by lifelong antigenic burden. Cells of the immune system are constantly exposed to a variety of stressors capable of inducing apoptosis, including antigens and reactive oxygen species continuously produced during immune response and metabolic pathways. The overall homeostasis of the immune system is based on the balance between antigenic load, oxidative stress, and apoptotic processes on one side, and the regenerative potential and renewal of the immune system on the other. Zinc is an essential trace element playing a central role on the immune function, being involved in many cellular processes, such as cell death and proliferation, as cofactor of enzymes, nuclear factors and hormones. In this context, the age associated changes in the immune system may be in part due to zinc deficiency, often observed in aged subjects and able to induce impairment of several immune functions. Thus, the aim of this work was to investigate the role of zinc in two essential events for immunity during aging, i.e. apoptosis and cell proliferation. Spontaneous and oxidative stress-induced apoptosis were evaluated by flow cytometry in presence of a physiological concentration of zinc in vitro on peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects of different age: a group of young subjects, a group of old subjects and a group of nonagenarians. In addition, cell cycle phases were analyzed by flow cytometry in PBMCs, obtained from the subjects of the same groups in presence of different concentration of zinc. We also analyzed the influence of zinc in these processes in relation to p53 codon 72 polymorphism, known to affect apoptosis and cell cycle in age-dependent manner. Zinc significantly reduces spontaneous apoptosis in all age-groups; while it significantly increases oxidative stress-induced late apoptosis/necrosis in old and nonagenarians subjects. Some factors involved in the apoptotic pathway were studied and a zinc effect on mitochondrial membrane depolarization, cytochrome C release, caspase-3 activation, PARP cleavage and Bcl-2 expression was found. In conclusion, zinc inhibits spontaneous apoptosis in PBMCs contrasting the harmful effects due to the cellular culture conditions. On the other hand, zinc is able to increase toxicity and induce cell death in PBMCs from aged subjects when cells are exposed to stressing agents that compromise antioxidant cellular systems. Concerning the relationship between the susceptibility to apoptosis and p53 codon 72 genotype, zinc seems to affect apoptosis only in PBMCs from Pro- people suggesting a role of this ion in strengthening the mechanism responsible of the higher propensity of Pro- towards apoptosis. Regarding cell cycle, high doses of zinc could have a role in the progression of cells from G1 to S phase and from S to G2/M phase. These effect seems depend on the age of the donor but seems to be unrelated to p53 codon 72 genotype. In order to investigate the effect of an in vivo zinc supplementation on apoptosis and cell cycle, PBMCs from a group of aged subjects were studied before and after six weeks of oral zinc supplementation. Zinc supplementation reduces spontaneous apoptosis and it strongly reduces oxidative stress-induced apoptosis. On the contrary, no effect of zinc was observed on cell cycle. Therefore, it’s clear that in vitro and in vivo zinc supplementation have different effects on apoptosis and cell cycle in PBMCs from aged subjects. Further experiments and clinical trials are necessary to clarify the real effect of an in vivo zinc supplementation because this preliminary data could encourage the of this element in all that disease with oxidative stress pathogenesis. Moreover, the expression of metallothioneins (MTs), proteins well known for their zinc-binding ability and involved in many cellular processes, i.e. apoptosis, metal ions detoxification, oxidative stress, differentiation, was evaluated in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from young and old healthy subjects in presence of different concentration of zinc in vitro. Literature data reported that during ageing the levels of these proteins increase and concomitantly they lose the ability to release zinc. This fact induce a down-regulation of many biological functions related to zinc, such as metabolism, gene expression and signal transduction. Therefore, these proteins may turn from protective in young-adult age to harmful agents for the immune function in ageing following the concept that several genes/proteins that increase fitness early in life may have negative effects later in life: named “Antagonistic Pleyotropy Theory of Ageing”. Data obtained in this work indicate an higher and faster expression of MTs with lower doses of zinc in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from old subjects supporting the antagonistic pleiotropic role of these proteins.

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

Mechanismus der Apoptose, Gentoxizität und DNA-Reparatur in Herpesvirus-Thymidinkinase-exprimierenden Säugerzellen nach Behandlung mit Antiherpes-Virustatika vom Typ der Nukleosidanaloga

Um Cytotoxizität und Gentoxizität nukleosidischer Antiherpes-Virustatika zu untersuchen, wurden stabile CHO-Klone etabliert, die Thymidinkinase (TK) des Herpes simplex-Virus Typ 1 (HSV-TK) oder des Varicella zoster-Virus (VZV-TK) exprimieren. In HSV-TK-exprimierenden Zellen wurde das Purinanalogon Ganciclovir (GCV) effizient in die genomische DNA eingebaut, worauf in den nächsten Replikationsrunden DNA-Strangbrüche und Aberrationen entstehen und Apoptose ausgelöst wird. GCV-induzierte Apoptose wird hauptsächlich über den mitochondrialen Weg vermittelt, wobei das anti-apoptotische Protein Bcl-2 im Mittelpunkt steht. Nach GCV-Behandlung konnte eine Caspase-9-vermittelte post-translationale Spaltung von Bcl-2 nachgewiesen werden. Das 23 kDa-großes Bcl-2-Fragment wirkt im Gegensatz zum intakten Bcl-2-Protein pro-apoptotisch und verstärkt die Cytochrom C-Freisetzung und damit die Aktivierung der Caspase-9, die Bcl-2 spaltet, was zu einem positiven 'Amplifikationsloop' des mitochondrialen apoptotischen Weges führt. In weiteren Experimenten wurde gezeigt, daß in die DNA inkorporiertes GCV durch Basenexzisionsreparatur repariert wird, wobei die DNA-Polymerase ß eine entscheidende Rolle spielt. Diese Reparatur führte zu einer signifikanten Reduktion der Apoptose und Klastogenität und damit zur Resistenzsteigerung gegenüber GCV. In VZV-TK-exprimierenden Zellen wurde gezeigt, daß Brivudin (BVDU), gleichermaßen Apoptose und Nekrose induzierte. Für die BVDU-induzierte Cytotoxizität konnte die Hemmung der Thymidylatsynthetase als Ursache identifiziert werden. Im Gegensatz zur GCV-induzierten Apoptose war für die BVDU-induzierte Apoptose der Rezeptor (Fas/CD95/APO-1)-vermittelte Weg von vorrangiger Bedeutung.