QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ciani, E. Articles by Contestabile, A. Search for Related Content PubMed PubMed Citation Articles by Ciani, E. Articles by Contestabile, A. Social Bookmarking                         What's this?

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).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter