Intracellular Triggering of Fas Aggregation and Recruitment of Apoptotic Molecules into Fas-enriched Rafts in Selective Tumor Cell Apoptosis

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts

School-Based Cardiovascular Health Promotion in Adolescents: A Cluster Randomized Clinical Trial.

IMPORTANCE School-based interventions offer an opportunity for health promotion in adolescence. OBJECTIVE To assess the effect of 2 multicomponent educational health promotion strategies of differing duration and intensity on adolescents' cardiovascular health (CVH). DESIGN, SETTING, AND PARTICIPANTS The SI! Program for Secondary Schools is a 4-year cluster randomized clinical intervention trial conducted in 24 secondary schools from Barcelona and Madrid, Spain, from September 7, 2017, to July 31, 2021. Eligible participants were adolescents enrolled in the first grade of secondary school. INTERVENTIONS Schools and their participants were randomized to receive a health promotion intervention (SI! Program) over 4 school years (long-term intervention [LTI], 8 schools, 412 adolescents) or 2 school years (short-term intervention [STI], 8 schools, 504 adolescents) or to receive the standard curriculum (control, 8 schools, 441 adolescents). MAIN OUTCOME AND MEASURES The primary end point was the between-group difference at 2 and 4 years in the change from baseline of the overall CVH score, as defined by the American Heart Association (range, 0-14 points, with a higher score indicating a healthier CVH profile). Intervention effects were tested with multilevel mixed-effects models. A complete-case intention-to-treat analysis was performed as the primary analysis. RESULTS Of the randomized students, the study enrolled 1326 adolescents (684 [51.6%] boys, mean [SD] age, 12.5 [0.4] years at recruitment) with a study completion rate of 86.0%. Baseline overall CVH scores were 10.3 points in the LTI group, 10.6 points in the STI group, and 10.5 points in the control group. After 2 years, at halfway through the LTI and at the end of the STI, the difference in the CVH score change was 0.44 points (95% CI, 0.01-0.87; P = .04) between the LTI group and the control group and 0.18 points (95% CI, -0.25 to 0.61; P = .39) between the STI group and the control group. At 4 years, differences for the LTI and STI groups vs control were 0.12 points (LTI: 95% CI, -0.19 to 0.43; P = .42) and 0.13 points (STI: 95% CI, -0.17 to 0.44; P = .38). No adverse events were reported. CONCLUSIONS AND RELEVANCE Overall, the tested school-based health promotion strategies in this randomized clinical trial had a neutral effect on the CVH of the adolescents. Although there was evidence of a marginal beneficial effect at a point halfway through implementation in the LTI group, such a benefit was not noted at 4 years. Further research is warranted into the efficacy of school-based health promotion programs. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT03504059.This work was supported by the SHE Foundation-la Caixa Foundation (LCF/PR/CE16/ 10700001) and the Fundació la Marató de TV3 (369/C/2016). Dr Santos-Beneit is recipient of grant LCF/PR/MS19/12220001 funded by “la Caixa” Foundation (ID 100010434). Dr Tresserra-Rimbau is a Serra Húnter Fellow. Dr Laveriano-Santos is supported by the FI-SDUR (EMC/503/2021) grant from the Generalitat de Catalunya. Mr Martínez-Gómez was a postgraduate fellow of the Ministerio de Ciencia e Innovación at the Residencia de Estudiantes (2020-2022) and is a recipient of grant FPU21/04891 (Ayudas para la formación de profesorado universitario, FPU-2021) from the Ministerio de Educación, Cultura y Deporte. Dr Álvarez-Benavides is a María Zambrano fellow. Dr Fernández-Jiménez is recipient of grants PI19/01704 and PI22/01560 funded by the ISCIII and cofunded by the European Union. Support was also provided by the Ministerio de Ciencia, Innovación y Universidades (AEI/FEDER, UE, grant PID2020-114022RB-I00), and Generalitat de Catalunya. The Institute for Nutrition and Food Safety Research (INSA-UB) is a Unit of Excellence (María de Maeztu CEX2021-001234-M). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the ISCIII, the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).S

LAL Regulators SCO0877 and SCO7173 as Pleiotropic Modulators of Phosphate Starvation Response and Actinorhodin Biosynthesis in Streptomyces coelicolor

LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism

Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells

Neutrophils possess a very short life-span, dying by apoptosis. HL-60 cells undergo apoptosis after neutrophil differentiation with dimethyl sulfoxide (DMSO). We have found that the onset of apoptosis in neutrophil-differentiating HL-60 cells correlates with the achievement of an apoptosis-related gene expression pattern similar to that of peripheral blood mature neutrophils. Using reverse transcriptase-polymerase chain reaction, cloning, and sequencing techniques, we have found that HL-60 cells express bak, bik, bax, bad, bcl-2, bcl-x(L), bcl-w, bfl-1, fas, and caspases 1-4 and 7-10. After DMSO treatment, bak, bcl-w, bfl-1, fas, and caspases 1 and 9 were up-regulated, whereas bik, bcl-2, and caspases 2, 3, and 10 were down-regulated at different degrees, achieving mRNA expression levels that correlated with those detected in peripheral blood neutrophils. Caspase-2 mRNA and protein expression was drastically reduced after HL-60 cell differentiation, being absent in both HL-60-differentiated neutrophils and mature neutrophils, whereas caspase-3 and -10 mRNA and protein expression were diminished upon HL-60 cell differentiation until achieving the respective levels found in mature neutrophils. Bak and bfl-1 mRNA levels were largely increased during DMSO-induced differentiation of HL-60 cells, and these genes were the bcl-2 family members that were expressed most abundantly in mature neutrophils. Bcl-2 overexpression or caspase inhibition prevented differentiation-induced apoptosis in HL-60 cells, but not their differentiation capability. Neutrophil spontaneous apoptosis was also blocked by the caspase inhibitor z-Asp-2,6-dichlorobenzoyloxymethylketone. Peripheral blood neutrophils expressed bak, bad, bcl-w, bfl-1, fas, and caspases 1, 3, 4, and 7-10, but hardly expressed bcl-2, bcl-x(L), bik, bax, and caspase-2. These results suggest that the above gene expression changes in neutrophil-differentiating HL-60 cells may play a role in the acquisition of the neutrophil apoptotic features.This research was supported in part by grant PB95-0713 from the Dirección General de Investigación Científica y Técnica (DGICYT), grant 1FD97-0622 from European Commission and Comisión Interministerial de Ciencia y Tecnología (CICYT), and grant VA32/99 from Junta de Castilla y León. A. M. S-B. is recipient of a fellowship from the Ministerio de Educación y Cultura of Spain.Peer Reviewe

Involvement of mitochondria and caspase-3 in ET-18-OCH3-induced apoptosis of human leukemic cells

The induction of cell death in leukemic HL-60 cells by the ether lipid I-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH3 induced disruption of the mitochondrial transmembrane potential (ΔΨ(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH3 also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH3 analogues unable to induce apoptosis failed to disrupt ΔΨ(m) and to activate caspase-3. ET-18-OCH3-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo ΔΨ(m) disruption upon ET-18-OCH3 incubation. Cyclosporin A partially inhibited ΔΨ(m) dissipation, caspase activation and apoptosis in ET-18-OCH3-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented ΔΨ(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH3-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone prevented ET-18-OCH3- induced PARP proteolysis and DNA fragmentation, but not ΔΨ(m) dissipation. ET-18-OCH3 did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH3-induced apoptosis did not require protein synthesis. Our data indicate that ΔΨ(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET.18.OCH3, and that the sequence of events in the apoptotic action of ET- 18-OCH3 on human leukemic cells is: ΔΨ(m) disruption, caspase-3 activation and internucleosomal DNA degradation.Grant sponsor: INKEYSA and Ministerio de Industria y Energía of Spain; Grant number: CDTI 97-0355; Grant sponsor: European Commission and Comisión Interminisiterial de Ciencia y Tecnología (CICYT); Grant number 1FD97-0622; Grant sponsor: Junta de Castilla y León; Grant number: VA32/99; Grant sponsor: CICYT; Grant number: SAF98/0047; Grant sponsor: Dirección General de Investigación Científica y Técnica (DGICYT); Grant number: PB95-0713; Grant sponsor: Fondo de Investigación Sanitaria (FIS96/1434).Peer Reviewe

Nonlinear optical 3-dimensional method for quantifying atherosclerosis burden

Changes in atherosclerosis burden provide an experimental measure of the effectiveness of therapeutic strategies. Unfortunately, none of the methods used in mice to assess atherosclerosis burden ex vivo is sufficiently accurate and fast enough to process the number of samples required in preclinical studies. We aimed to design an easy-to-implement and relatively fast method for accurate volumetric quantification of atheroma plaque burden in mice.Dr Redondo is supported by the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2009-10708 and SAF2012-34296) and Fundación La Marató TV3 (264/C/2012). The Red de Investigación Cardiovascular of the Spanish Ministry of Health (Ministerio de Sanidad) supports Dr Redondo (RD12/0042/0022) and Dr Jiménez-Borreguero (RD12/0042/0056). Dr Campanero was supported by MINECO (SAF2010-15126). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation. N. Méndez-Barbero holds a Formacion del Profesorado Universitario fellowship (FPU2008-1500).Peer Reviewe