Research Article
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Nitric oxide negatively regulates proliferation and promotes neuronal differentiation through N-Myc downregulation Elisabetta Ciani1,*, Sabina Severi1, Andrea Contestabile1, Renata Bartesaghi1 and Antonio Contestabile2 1Department 2Department
of Human and General Physiology, University of Bologna, Piazza di Porta San Donato 2, 40127, Bologna, Italy of Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy
*Author for correspondence (e-mail:
[email protected])
Accepted 7 June 2004 Journal of Cell Science 117, 4727-4737 Published by The Company of Biologists 2004 doi:10.1242/jcs.01348
Summary Nitric oxide (NO) has been found to act as an important negative regulator of cell proliferation in several systems. We report here that NO negatively regulates proliferation of neuronal cell precursors and promotes their differentiation by downregulating the oncogene N-Myc. We have studied this regulatory function of NO in neuroblastoma cell lines (SK-N-BE) and in primary cerebellar granule cell cultures. In a neuronal NO synthase (nNOS) overexpressing neuroblastoma cell line exposed to the differentiative action of retinoic acid, NO slowed down proliferation and accelerated differentiation towards a neuronal phenotype. This effect was accompanied by a parallel decrease of N-Myc expression. Similar results could be obtained in parental SK-N-BE cells by providing an exogenous source of NO. Pharmacological controls demonstrated that NO’s regulatory actions on cell proliferation and N-Myc expression were mediated by
Key words: nNOS, Neuroblastoma cells, Cerebellar granule cells, Development, N-Myc, Neurogenesis
Introduction New functions played by nitric oxide (NO) as an intercellular and intracellular signaling molecule in the brain are continuously demonstrated and increasing evidence suggests that NO might be a primary player in the program of neurogenesis and neuronal differentiation (Bredt and Snyder, 1994a; Contestabile et al., 2003; Packer et al., 2003). NO synthase (NOS) expression and NO synthesis are strongly induced in cultured cells treated with growth factors, and many of the pleiotropic effects of growth factors might be mediated through NO (Heneka et al., 1998; Obregon et al., 1997; Peunova and Enikolopov, 1995; Poluha et al., 1997). Moreover, neuronal NOS (nNOS), the major NOS isoform in the mammalian brain, is transiently expressed in the developing brain in a pattern suggesting its involvement in neural development (Bredt and Snyder, 1994b). Furthermore, the ability of NO reversibly to suppress cell division (Garg and Hassid, 1990; Lepoivre et al., 1990), coupled with its ability to regulate gene expression (Hemish et al., 2003), has been recently exploited in several neural developmental models (Champlin and Truman, 2000; Enikolopov et al., 1999; Peunova and Enikolopov, 1995; Peunova et al., 2001; Poluha et al., 1997). These researches have highlighted a primary role
for NO as a negative regulator of neural cells proliferation and the molecular bases for its antiproliferative action are beginning to be understood. NO can regulate gene expression by modulating transcription factors or the translation or stability of mRNA, or by modifying proteins, mainly acting through the cGMP messenger system and/or direct chemical interactions with macromolecules (Bogdan, 2001; Marshall et al., 2000; Schindler and Bogdan, 2001). In Drosophila, for instance, the control of cell division is mediated by direct interaction with the retinoblastoma (Rb) pathway, and it has been proposed that this might occur through S-nitrosylation of Rb (Kuzin et al., 2000). N-Myc is a member of the Myc proto-oncogene family, which also includes c-Myc and L-Myc. Myc proteins are known to promote proliferation and/or transformation in many cell types (Henriksson and Luscher, 1996), and their loss results in severely compromised cell cycle regulation (Mateyak et al., 1997). N-Myc oncogene amplification and/or expression play an important role in neuroblastoma tumor aggressiveness. Amplification of N-Myc expression indeed occurs in about 30% of neuroblastomas, it is associated with rapid tumor progression and survival expectancy less than 15% (Brodeur et al., 1984; Westermann and Schwab, 2002). Furthermore, during retinoic acid (RA) induced differentiation
cGMP as an intermediate messenger. Furthermore, NO was found to modulate the transcriptional activity of NMyc gene promoter by acting on the E2F regulatory region, possibly through the control of Rb phosphorylation state, that we found to be negatively regulated by NO. In cerebellar granule cell cultures, NOS inhibition increased the division rate of neuronal precursors, in parallel with augmented N-Myc expression. Because a high N-Myc expression level is essential for neuroblastoma progression as well as for proliferation of neuronal precursors, its negative regulation by NO highlights a novel physiopathological function of this important messenger molecule.
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of neuroblastoma cells in vitro, N-Myc undergoes transcriptionally mediated downregulation, in parallel with decreased cell proliferation, which is felt to be a key event in the differentiation process (Bordow et al., 1998; Chan et al., 1997; Seeger et al., 1985; Thiele and Israel, 1988; Thiele et al., 1985). Thus, Myc proteins are involved in fundamental cellular processes including cell proliferation, survival/apoptosis and differentiation, through activation or repression of specific sets of target genes (Dang, 1999; Eisenman, 2001; Grandori et al., 2000). The role of Myc proteins in the regulation of cell proliferation is consistent with studies in Drosophila (Johnston et al., 1999) as well as with the effects of their overexpression in mammalian cells (Beier et al., 2000; Iritani and Eisenman, 1999; Kim et al., 2000; Schuhmacher et al., 1999). Concerning neural cells, a recent study has demonstrated that N-Myc is required for brain development and that targeted loss of N-Myc function specifically disrupts the ability of neuronal progenitor cells to expand, differentiate, and populate the brain (Knoepfler et al., 2002). Regulation of Myc gene expression has been shown to occur at multiple levels, including gene transcription (Park and Wei, 2003), premature termination of translation (Krumm et al., 1992; Perez-Juste et al., 2000) and translocation (Kanda et al., 2000; Ratsch et al., 2002). On the basis of the literature data surveyed above, negative regulation of N-Myc expression is a plausible mediator of the antiproliferative action of NO. However, no research has yet been performed to address this possible link. We report here for the first time that the negative regulation of proliferation exerted by NO on neuroblastoma cells is paralleled by downregulation of N-Myc expression. Furthermore, we provide evidence that the same holds true also for neuronal precursors in primary cultures of cerebellar granule cells (CGCs). Finally, we suggest that the cellular pathway linking NO to N-Myc downregulation involves transcriptional regulation of N-Myc expression by the Rb-E2F system. Materials and Methods Cell culture The human neuroblastoma SK-N-BE cells (Biedler et al., 1978) were seeded at a density of 105 cells cm–2 on plastic culture plates and were grown to confluence in RPMI 1640 medium (Gibco BRL) containing 10% heat-inactivated fetal calf serum (FCS) (Gibco BRL), 2 mM glutamine, 100 units ml–1 penicillin and 50 µg ml–1 streptomycin (Sigma) at 37°C in a 5% CO2 humidified atmosphere. Upon reaching confluence, the cells were dispersed with trypsin, split and subcultured. Cultures were routinely observed under a phase-contrast inverted microscope and under fluorescence microscope after vital staining with fluorescein diacetate. Primary cultures of CGCs were prepared from the cerebella of 7day-old Wistar rat pups as previously described (Gallo et al., 1982). Briefly, cerebella were removed and dissected from their meninges in Krebs’ buffer (120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, 10 mM glucose pH 7.4) containing 0.3% bovine serum albumin (BSA). Tissue was dissociated with trypsin at 37°C for 15 minutes and triturated 15 times using a Pasteur pipette in a DNase/soybean-trypsininhibitor solution. The cells were plated at a density of 2.4×106 per 35 mm dish previously coated with poly-L-lysine and maintained in basal modified Eagle’s (BME) medium (Gibco) containing 10% FCS, 2 mM glutamine, 100 µg ml–1 gentamicin and 25 mM KCl.
Treatments All-trans retinoic acid was dissolved in dimethyl sulfoxide (DMSO) at a 10 mM concentration and stored in the dark at –20°C. This stock was added to culture flasks of neuroblastoma cells to a final 10 µM concentration. Control flasks were treated with an equivalent volume of DMSO solvent alone. The various pharmacological agents tested (L-NAME, DETA NONOate, ODQ, 8Brc-GMP) were added in a volume of 1 µl ml–1 in the medium at the concentrations and for the times specified in the figure legends. Measurements of neurite growth Cells were fixed in 4% paraformaldehyde, 4% sucrose at 37°C, and were stained using hematoxylin and eosin. Cells possessing one or more neurites longer than the diameter of the cell bodies were considered to be positive for counting. The neurite growth index (NGI) (Image pro plus software) was used to obtain an overall measurement of neurite length. Images of the treated cells were captured with a digital camera. All neurites in each region of interest (ROI) were manually traced and the length was measured using Image Pro Plus (Scanalytics, Fairfax, VA, USA). The NGI was calculated by dividing the sum of neurite length by the number of cells in each ROI. For each experiment, at least 150 cells were measured. Proliferation assay Cell proliferation was determined using a bromodeoxyuridine (BrdU) labeling and detection kit III (Roche, Mannheim, Germany) following the instruction manual. Briefly, cells were cultured in a microtiter plate (96 wells) and BrdU was added to the culture medium for 2 hours to be incorporated into newly synthesized DNA. Following fixation of cells, cellular DNA was partially digested by nuclease. Next, a peroxidase (POD) labeled antibody to BrdU (antiBrdU POD) was added. The absorbance of the sample was determined in a microtiter plate reader at 405 nm with a reference wavelength of 490 nm. Ornithine-decarboxylase assay Ornithine decarboxylase (ODC) was measured in supernatants of cell homogenates, centrifuged at 10,000 g for 15 minutes at 4°C using a radiochemical method (Baudry et al., 1986). Luciferase measurement Transient transfection experiments were performed using polyethylenimine (PEI 25K) as DNA carrier according to the previously described method (Bunone et al., 1997). Plasmids used for transfection were: CMVLuc, rsvSP1, N-MycLuc and NMyc∆E2FLuc. 24 hours after transfection, cells were washed twice with ice-cold PBS and lysed by incubation in 50 mM Tris-MES (pH 7.8) (Sigma), 1 mM dithiothreitol (DTT) and 1% Triton X-100 for 5 minutes on ice. The lysate was cleared of cellular debris by centrifugation (de Wet et al., 1987). Luciferase assays were performed with a TD-20/20 luminometer (Promega, Madison, WI). Determination of nitrite and nitrate concentrations Nitrite, a stable product of NO oxidation, plus nitrates previously converted to nitrites (Miranda et al., 2001), were determined by measuring accumulation in culture medium through the Griess reaction as described previously (Dong et al., 1994). In brief, culture media (100 µl) were collected, mixed with 100 µl vanadium (III) chloride (400 mg per 50 ml 1 M HCl), added with 50 µl of sulfanilamide (dissolved in 1.2 M HCl) and 50 µl Nnaphthylethylenediamine dihydrochloride. After 15 minutes at room temperature, samples were measured at a wavelength of 540
NO downregulates N-Myc nm. Nitrite concentrations were calculated against a NaNO2 standard. Apoptosis assay A sandwich enzyme-linked immunosorbent assay (ELISA) method was used to assess apoptosis (Cell Death ELISA; Roche Biochemicals). The assay measures the enrichment of histoneassociated DNA fragments in apoptotic cells. Detection of bound nucleosomes from the samples is made using a monoclonal anti-DNA antibody with a POD label. Bound anti-DNA-POD is quantified using the POD substrate 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS), whose product is measured by absorbance at 405 nm. Northern analysis Approximately 30 µg total RNA per sample were loaded per lane and fractionated on a 1% agarose formaldehyde gel. Following transfer to Hybond N membrane (Amersham International, Wiltshire, UK), NMyc mRNA was detected using an α32P-labeled full-length probe. Cyclophilin probes were used for quantification as described previously (Taubenfeld et al., 2001). Membranes were washed to a maximum stringency of 0.1× sodium chloride-sodium citrate (SSC) buffer, 0.1% sodium dodecyl sulfate (SDS) at 65°C. Autoradiography was performed using Kodak XAR film at –70°C with an intensifying screen. To measure RNA half-life, cells were exposed to actinomycin D at 10 µg ml–1 on day 2 of treatment with RA or solvent control. RNA was harvested from treated and control cells after 0 minutes, 45 minutes, 90 minutes and 180 minutes of exposure to actinomycin D. The time course and intensity of such treatment has been shown to inhibit N-Myc RNA synthesis by >95% without significantly decreasing viability (data not shown). Once harvested, RNA was analysed by northern blotting as described above. The N-Myc message half-life was calculated from densitometric laser scans of autoradiographs using the signal intensity from cyclophilin-encoding mRNA as a standard. RT-PCR assays Total RNA, free from chromosomal DNA contamination, was isolated using Trizol according to the supplier’s instructions and reverse transcribed with SUPERSCRIPT II RNase H-Reverse transcriptase (Life Technologies) using customer-synthesized (Life Technologies) oligo-dT12-18 primers. Reverse-transcription polymerase chain reaction (RT-PCR) primers for glyceraldehyde-3-phosphate dehydrogenase (GADPH) and N-Myc are as follows. GADPH (451 bp) sense, 5′-ACCACAGTCCATGCCATCAC-3′, antisense, 5′TCCACCACCCTGTTGCTGTA-3′; N-Myc (500 bp) sense, 5′GTCACCACATTCACCATCACTGT-3′, antisense 5′-AGCGTGTTCAATTTTCTTTAGCA-3′. Briefly, 2 µg of total RNA was incubated for 45 minutes at 42°C in 20 µl containing 25 µM of the primer, 10 mM DTT, 0.5 mM dATP, dCTP, dGTP and dTTP, 200 units reverse transcriptase and 1× First Strand Buffer (Life Technologies). A 1 µl aliquot of reverse-transcribed cDNA was subjected to PCR in a 50 µl mixture containing 3′ and 5′ primers, each 2 µM. PCR amplification was conducted for 21 cycles (GADPH) or 28 cycles (NMyc) using the following conditions: denaturation, 40 seconds at 94°C; annealing, 30 seconds at 55°C (N-Myc) or 56°C (GADPH); primer extension, 2 minutes at 72°C. PCR products were electrophoresed on agarose gels containing ethidium bromide and visualized under ultraviolet light. Band intensities (arbitrary optical density units) were evaluated using a Bio-Imaging Analyzer system (Amersham) combined with densitometry. Intensities for amplified Bcl-2 were normalized against those obtained for GADPH in the same samples. The linearity of PCR amplification was verified by amplifying several dilutions of reverse-transcribed cDNA.
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Western blotting The following antibodies were used for analysis of nNOS (polyclonal antibody at 1:500), PCNA (polyclonal antibody at 1:500), N-Myc (monoclonal antibody at 1:250), Rb (polyclonal antibody at 1:500), phosphorylated Rb (P-Rb) (polyclonal antibody at 1:500) all from Santa Cruz Biotechnology and β-actin (polyclonal antibody at 1:1000; Sigma). Western blots were performed on 2×106 cells per experimental point. Cell pellets were lysed by homogenization at 4°C with a lysis buffer containing 1% deoxycholate, 1 µg ml–1 aprotinin, 2 µg ml–1 leupeptin, 1 mM phenylmethylsulfonyl fluoride and 1 mM sodium orthovanadate for 10 minutes. Lysates were immediately processed by western blot or kept frozen until assayed. Protein concentrations of samples were estimated by the Lowry method (Lowry et al., 1951). Equivalent (50 µg) amounts of proteins per sample were subjected to electrophoresis on a 10% SDSpolyacrylamide gel. The gel was then blotted onto a nitrocellulose membrane and equal loading of protein in each lane was assessed by brief staining of the blot with 0.1% Ponceau S. Blotted membranes were blocked for 1 hour in a 4% suspension of dried skimmed milk in PBS and incubated overnight at 4°C with: primary antibodies. Membranes were washed and incubated for 1 hour at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (dilution 1:1000). Specific reactions were revealed with the ECL western blotting detection reagent (Amersham, Piscataway, NJ.). Immunoprecipitation studies To prepare cell extracts, cells were washed three times with PBS and then lysed in RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate, 1× protease inhibitor mixture (Roche Applied Science), 1 mM sodium vanadate] for 15 minutes on ice. The lysates were centrifuged in an Eppendorf centrifuge at 10,000 g and 4°C for 15 minutes. Cell lysates containing an equal amount of protein (500 µg) were incubated with prewashed protein-G/Sepharose beads for 1 hour before incubation with primary antibody overnight at 4°C under gentle shaking. Prewashed Sepharose beads were further incubated with the lysateantibody mixture for 1-2 hours. Beads were washed twice in 200 mM NaCl, 1× RIPA, followed by a final wash in 1× RIPA. Proteins were resolved by 10% SDS polyacrylamide-gel electrophoresis (SDSPAGE) and subsequent western blot analysis. Immunocytochemistry To label cells in S phase in vitro, purified CGCs were incubated in 10 µM BrdU (Sigma) for 2 hours, 24 hours after plating. Then the cells were fixed in 70% ethanol in 50 mM glycine buffer, pH 2.0, at –20°C for 20 minutes. The cells were then incubated with an antiBrdU monoclonal antibody (diluted 1:100; Chemicon), followed by an avidin-biotin amplified immunohistochemical method as described previously (Ciani and Contestabile, 1994). For doubleimmunofluorescence cytochemistry, cells were grown on coverslips, washed twice with PBS and fixed for 10 minutes with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (0.1 M NaH2PO4/ Na2HPO4) at pH 7.4. After washing with PBS, cells were permeabilized and treated with blocking solution (5% normal goat serum and 0.4% Triton X-100 in PBS) for 30 minutes. Cells were incubated overnight with anti-BrdU monoclonal antibody (diluted 1:100; Chemicon) and anti-glial-fibrillar-acidic-protein (GFAP) polyclonal antibody (dilution 1:800; Sigma). Coverslips were washed four times for 5 minutes each, with PBS/Triton X-100 solution (0.1% Triton X-100 in 0.1 M phosphate buffer 0.9% NaCl). Cells were incubated with Cy5-conjugated goat anti-rabbit secondary antibody (dilution 1:500; Sigma) and fluorescein-isothiocyanate-conjugated goat anti-mouse secondary antibody (dilution 1:250; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour. Coverslips were washed
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twice with PBS, mounted on glass slides, observed and photographed with a Zeiss fluorescence microscope. Statistics Data were expressed as means±s.e.m. and statistical significance was assessed by using one-way analysis of variance (ANOVA) followed by Bonferroni’s test (SigmaStat software). Differences were considered to be significant starting from P
