Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells

BACKGROUND: Autophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS. METHODS: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation. RESULTS: Autophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress. CONCLUSIONS: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress

Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34+ stem cells into the expanding tumor vasculature

The Ewing's sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF165), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34+ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34 + cells migrate from the bone marrow to Ewing's sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF165 in CD34+ cell migration. Human umbilical cord CD34+ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE-cadherin and mouse CD31, indicating involvement of donor-derived human cells in the tumor vessels. To determine the role of VEGF165 in the chemoattraction of CD34+ cells, we generated two VEGF 165-deficient TC71 clones, a stable anti-sense VEGF165 cell line (Clone 17) and a VEGF165 siRNA-inhibited clone (TC/siVEGF7-1). The resulting VEGF165-deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34+ cells into both VEGF165-deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewing's sarcoma and that VEGF165 is critical for the migration of CD34+ cells from the bone marrow into the tumor. © 2006 Wiley-Liss, Inc.Fil: Lee, Tim H.. University of Texas; Estados UnidosFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. University of Texas; Estados UnidosFil: Worth, Laura L.. University of Texas; Estados UnidosFil: Guan, Hui. University of Texas; Estados UnidosFil: Ellis, Lee M.. University of Texas; Estados UnidosFil: Kleinerman, Eugenie S.. University of Texas; Estados Unido

Residential mobility and childhood leukemia.

AimsStudies of environmental exposures and childhood leukemia studies do not usually account for residential mobility. Yet, in addition to being a potential risk factor, mobility can induce selection bias, confounding, or measurement error in such studies. Using data collected for California Powerline Study (CAPS), we attempt to disentangle the effect of mobility.MethodsWe analyzed data from a population-based case-control study of childhood leukemia using cases who were born in California and diagnosed between 1988 and 2008 and birth certificate controls. We used stratified logistic regression, case-only analysis, and propensity-score adjustments to assess predictors of residential mobility between birth and diagnosis, and account for potential confounding due to residential mobility.ResultsChildren who moved tended to be older, lived in housing other than single-family homes, had younger mothers and fewer siblings, and were of lower socioeconomic status. Odds ratios for leukemia among non-movers living <50 meters (m) from a 200+ kilovolt line (OR: 1.62; 95% CI: 0.72-3.65) and for calculated fields ≥ 0.4 microTesla (OR: 1.71; 95% CI: 0.65-4.52) were slightly higher than previously reported overall results. Adjustments for propensity scores based on all variables predictive of mobility, including dwelling type, increased odds ratios for leukemia to 2.61 (95% CI: 1.76-3.86) for living < 50 m from a 200 + kilovolt line and to 1.98 (1.11-3.52) for calculated fields. Individual or propensity-score adjustments for all variables, except dwelling type, did not materially change the estimates of power line exposures on childhood leukemia.ConclusionThe residential mobility of childhood leukemia cases varied by several sociodemographic characteristics, but not by the distance to the nearest power line or calculated magnetic fields. Mobility appears to be an unlikely explanation for the associations observed between power lines exposure and childhood leukemia

Extraction of Boron Nitride Nanotubes and Fabrication of Macroscopic Articles Using Chlorosulfonic Acid

Due to recent advances in high-throughput synthesis, research on boron nitride nanotubes (BNNTs) is moving towards applications. One future goal is the assembly of macroscopic articles of high aspect ratio, pristine BNNTs. However, these articles are presently unattainable because of insufficient purification and fabrication methods. We introduce a solution process for extracting BNNTs from synthesis impurities without sonication or the use of surfactants and proceed to convert the extracted BNNTs into thin films. The solution process can also be used to convert assynthesized materialwhich contains significant amounts of hexagonal boron nitride (h-BN) into mats and foams with controllable structure and dimension. The scalable solution extraction method, combined with further advances in synthesis and purification, contributes to the development of all-BNNT macroscopic articles, such as fibers and 3-D structures

Incidencias en Trichostrongylídeos en relación con los patrones de remolinos frontales en bovinos

La transmisión de los nematodes gastrointestinales trichostrongylideos de los rumiantes en pastoreo está asociada con la ingestión de sus larvas infectivas distribuidas en las pasturas. Con el objetivo de analizar si la incidencia de estas parasitosis en bovinos podría estar influenciada por la posición de los remolinos pilosos de la cabeza, fueron evaluadas las intensidades medias e Intervalos de confianza 95 % de los recuentos de huevos de nematodes por gramo de materia fecal (hpg) de 126 muestras pertenecientes a tres rodeos de bovinos Aberdeen Angus de 8 a 10 meses de edad. Los valores hallados para los animales con remolinos-altos fueron de 203.7 (155.6-259.3), 300.1 (200-440.1) y 360 (200-520), para animales con los remolinos-medios de 253.4 (173.3-360.1), 417 (216.6-600.7) y 416.7 (341.67-500) y para los animales con remolinos-bajos de 250 (186.4-318.2), 362.8 (212.5-525.5) y 433.6 (350.06-522.6) en los rodeos denominados Villegas, Zavalla y Tierra. En los tres rodeos, los bovinos con remolinos medios y bajos presentaron tendencias mayores en sus incidencias medias, mientras que en los animales con remolinos pilosos altos, las incidencias medias tendieron a ser más bajas. Otros factores, además del comportamiento, pueden influir en las intensidades del parasitismo por nematodes trichostrongylideos según los tipos de remolinos. Entender las diferencias probablemente esté asociado a explicar cómo se conforma el estilo de pastoreo en los grupos de animales en relación a cada subtipo en la zona de ingesta.Transmission of gastrointestinal nematodes from grazing ruminants is associated with the ingestion of their infective larvae distributed in pastures. In order to analyze if the incidence of parasitism in cattle could be influenced by associated to hair whorl position of the head, the mean intensities and 95% confidence intervals of nematode egg counts were evaluated by gram of fecal material (epg) of 126 samples cattle of 8 to 10 months old in three ranchs of Aberdeen Angus. The values were for animals with high whorl 203.7 (155.6-259.3), 300.1 (200-440.1) and 360 (200-520), for animals with middle whorl 253.4 (173.3-360.1), 417 (216.6)-600.7) and 416.7 (341.67-500), and for animals with low whorl 250 (186.4-318.2), 362.8 (212.5-525.5) and 433.6 (350.06-522.6) in the herds Villegas, Zavalla and Tierra. In the three rodeos, the animals with medium and low whorl presented higher trends in their mean incidences, while in animals with high hair whorl, the average incidences tended to be lower. Other factors, besides behavior, can influence the intensities of parasitism by trichostrongylid nematodes according to the types of swirls. Understanding the differences is probably associated with explaining how the grazing style is conformed in the groups of animals in relation to each subtype in the intake zone.EEA General VillegasFil: Ardusso, Gerardo L. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Lacovara, Martin Andres. Becario INTA-AUDEAS CONADEV; ArgentinaFil: Graziati, Georgina. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Angulo Lewille, M. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; ArgentinaFil: Kleinerman, Gabriela. The Hebrew University of Jerusalem. Koret School of Veterinary Medicine; IsraelFil: Buffarini, Miguel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria General Villegas; ArgentinaFil: Giudici, Claudio. Universidad Nacional de Rosario. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Mssbauer study of Се2Fe17 compound in different magnetic states

Mssbauer spectra of two samples of the Ce2Fe17 compound have been analyzed and hyperfine parameters, compared. The samples were subjected to different preparation techniques and display different magnetic properties. It is established that to well fit the Mssbauer spectra of these samples, an appropriate model should be used that takes into account an additional subspectrum differing in hyperfine parameters from the subspectra employed in conventional models. It is shown that in the ferromagnetic state, the samples contain local regions with an antiferromagnetic order, the volume fraction of which, being different for two samples, increases on approaching the temperature of ferro-to-antiferromagnet transition