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First published online 25 August 2004
doi: 10.1242/jcs.01348

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Nitric oxide negatively regulates proliferation and promotes neuronal differentiation through N-Myc downregulation

¹ Department of Human and General Physiology, University of Bologna, Piazza di Porta San Donato 2, 40127, Bologna, Italy
² Department of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy

View larger version (16K): [in a new window]   Fig. 1. (A) nNOS expression in parental SK-N-BE, clone-4 and pool of all the selected clone cells by immunoblot analysis. (B) Induction of nNOS expression in RA-treated parental SK-N-BE, clone-4 and pooled clone cells. The expression of nNOS was determined by western-blot analysis. Cells were exposed to 10 µM RA for a total of 10 days. 50 µg protein were subjected to SDS-PAGE and the blot was probed with anti-nNOS antibody. (C) Catalytic NOS activity evaluated through nitrite-plus-nitrate accumulation in the medium, expressed as a proportion of the activity in control cultures without RA (the absolute concentration of the control is 1.5 µM). Bars are the mean±s.e.m. of three independent experiments performed in duplicate. *, P<0.01 (Bonferroni's test after ANOVA).

View larger version (47K): [in a new window]   Fig. 2. (A) Parental and clone-4 cells stained with hematoxylin-eosin before (a,b) and after (c,d) 48 hours of treatment with 10 µM RA. Notice the more differentiated neuronal phenotype acquired by clone-4 cells after such a short exposure to RA (d). (B) Effects of RA exposure on neurite length in parental and clone-4 cells. Notice the large increase in neurite length with 2 days of exposure to RA in clone-4 and parental cells co-treated with RA and 200 µM DETA-NONOate. Bars are the mean±s.e.m. from measuring neurite extension of at least 150 cells per dish in three separate dishes. (C, left) In parallel to stimulation of clone-4 differentiation, RA also slowed down cell proliferation in this clone compared with parental cells, an effect completely reversed by blocking NOS activity using L-NAME (1-5 mM) or the guanylate cyclase inhibitor ODQ (50 µM). (C, right) Inhibition of BrdU incorporation in SK-N-BE cells by RA and DETA-NONOate (50-200 µM) or 8Br-cGMP (250 µM). The antiproliferative effects were determined after treating the parental and clone-4 cells for 2 days with 10 µM RA and labeling with BrdU (10 µM) for the last 2 hours. Results are expressed as the percentage BrdU incorporation relative to parental cells treated with RA. Bars are the mean±s.e.m. of four experiments. *, P<0.01 compared with control cultures; #, P<0.01 compared with clone 4 (Bonferroni's test after ANOVA).

View larger version (19K): [in a new window]   Fig. 3. (A) Effect of RA treatments on PCNA expression in parental and nNOS overexpressing SK-N-BE cells. Levels of PCNA were visualized by western blot. (B) Effect of RA treatments on ODC activity in parental and clone-4 cells. Bars are the mean±s.e.m. of three experiments. *, P<0.01 compared with control (Bonferroni's test after ANOVA). (C) Quantification of cell death after 1-7 days RA exposure by a specific ELISA kit. Bars are the mean±s.e.m. of four experiments. *, P<0.01 compared with clone 4 without RA; #, P<0.01 compared with parental cells without RA (Bonferroni's test after ANOVA).

View larger version (20K): [in a new window]   Fig. 4. (A) Effect of RA treatment on N-Myc expression in parental and nNOS overexpressing cells (clone 4 and pooled clones). Levels of N-Myc were evaluated by western blot. (B) N-Myc was quantified through its ratio to actin content of the samples. Bars are the mean±s.e.m. of four experiments. *, P<0.01 compared with control (Bonferroni's test after ANOVA). (C) Effect of RA treatment on N-Myc in clone 4 after the addition of the NOS inhibitor L-NAME (1-5 mM) and the guanylate cyclase inhibitor ODQ (50 µM) to the culture medium. (D) The negative regulation of N-Myc protein expression was replicated in parental cells using increasing concentrations of DETA NONOate (50-200 µM) and the cGMP analog 8Br-cGMP (250 µM).

View larger version (17K): [in a new window]   Fig. 5. (A) N-Myc mRNA half-life. The intensity of the N-Myc mRNA signal was measured by laser densitometry of autoradiographs from northern blots and plotted as a function of time after exposure to actinomycin D. Parental and clone-4 cells treated with RA did not show alteration in the half-life of N-Myc mRNA. (B) Effect of RA treatment on luciferase activity in parental and clone-4 cells transfected with N-MycLuc and N-MycE2FLuc (3 µg). 12 hours after transfection, cells were exposed to RA for 24 hours. Luciferase activity is given as a percentage of the respective control condition in the absence of RA (hatched line) for each transfection condition. Bars are the mean±s.e.m. of four experiments. *, P<0.01 compared with control (Bonferroni's test after ANOVA). (C) DETA-NONOate dose-dependently decreased the activity of N-MycLuc reporter transiently transfected in parental cells. *, P<0.01 compared with control (Bonferroni's test after ANOVA).

View larger version (22K): [in a new window]   Fig. 6. Alterations in Rb phosphorylation in parental and clone-4 cells exposed to RA. (A) Total cell lysates from parental or clone-4 cells were immunoprecipitated with an anti-Rb antibody and western blotted with an anti-phospho-Rb monoclonal antibody. (B) Immunoprecipitate densitometric analysis of the proportion of PRb to total Rb from parental cells treated with RA and the NO donor DETA-NONOate (200 µM) or the cGMP analog 8Br-cGMP (250 µM), and from clone-4 cells treated with RA and L-NAME (5 mM). Bars are means±s.e.m. of four experiments. *, P<0.01 compared with control (Bonferroni's test after ANOVA).

View larger version (36K): [in a new window]   Fig. 7. (A) Phase-contrast photographs of CGCs after BrdU immunocytochemistry. Arrows point to BrdU labeled cells. (B) L-NAME and Shh stimulate BrdU incorporation in CGC cultures: quantification of BrdU labeled cells. Cells from neonatal rat cerebellum were cultured for 1 day in control medium or supplemented with 500 µM L-NAME or 3 µg ml-1 Shh. Cultures were pulsed with 10 µM BrdU for the final 4 hours and processed for BrdU immunocytochemistry and subsequent quantification. Bars are the mean±s.e.m. of four experiments. *, P<0.01 compared with control (Bonferroni's test after ANOVA). (C) Semiquantitative RT-PCR analysis was performed with RNA isolated from CGCs treated with L-NAME or Shh for 24 hours. Amplification of the 500 bp PCR product represents N-Myc mRNA. Bars are the mean±s.e.m. of four experiments. *, P<0.01 (Bonferroni's test after ANOVA). (D) Double-immunofluorescence cytochemistry with anti-BrdU and anti-GFAP antibodies of CGCs cultured for 1 day in control medium or supplemented with 500 µM L-NAME. Images of immunoreactivity (left) for BrdU (green), GFAP (red) and both markers (yellow) in CGC cultures. The ratio of total BrdU-labeled cells on BrdU+GFAP-positive cells was quantified in control and compared with L-NAME-treated cultures (right). Bars are the mean±s.e.m. of three experiments. *, P<0.01 (Bonferroni's test after ANOVA).