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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00834

Journal of Cell Science 117, 293-301 (2004)
Published by The Company of Biologists 2004
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Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line

David Goldschneider1, Etienne Blanc1, Gilda Raguénez1, Michel Barrois3, Agnès Legrand2, Gwenaëlle Le Roux1, Hedi Haddada2, Jean Bénard1,3 and Sétha Douc-Rasy1,*

1 Centre National de la Recherche Scientifique-Unité Mixte de Recherche 8126, 39 rue Camille Desmoulins, 94805 Villejuif, France
2 Institut National de la Santé et de la Recherche Médicale U 362, 39 rue Camille Desmoulins, 94805 Villejuif, France
3 Laboratoire de Génétique, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France



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  		Fig. 1. Identification of the p53 mutation by insertion in the IGR-N-91 cell line. SH refers to SH-SY5Y cells; IGR refers to IGR-N-91 cells. (A) Western blot (left panel) of protein total lysates showing that the p53 protein band is of a higher molecular mass in IGR cells than in SH cells. Electrophoresis (right panel), in 2% agarose, of RTPCR products spanning exons 1-11, show an amplified fragment in IGR approximately 300 bp longer than in SH. (B) Restriction endonuclease fragments from StyI, RsaI and TaqI. (C) Polyacrylamide electrophoresis of exons 6-10 showing a higher band (~300 bp) in IGR than in SH (left panel); when PCR was performed using the forward primer, spanning the end of exon 9 and the first four nucleotides at the beginning of exon 7, only IGR had an amplified fragment (right panel). (D) Schematic representing the restriction sites of each enzyme in B. Arrowhead, IGR amplified fragment.

 


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  		Fig. 3. Induction of endogenous p53 and p21 proteins by p73 ectopic overexpression in SH-SY5Y and IGR-N-91 cells. (A) p53 and p21 proteins were detected by western blot analysis of total protein extracts; the blots were incubated with monoclonal anti-p53 antibody (upper panels) for p53 expression and with monoclonal anti-p21 antibody (middle panels) for p21 expression. Upregulation of p21 protein was observed only with Ad-TAp73{alpha} but not with Ad-{Delta}Np73{alpha}. (B) Northern blot showed no induction of p53 mRNA expression. Total RNA (10 µg) was prepared 48 hours after infection and submitted to northern blotting with p53 cDNA probe. Ethidium bromide staining of 28S and 18S rRNAs is used to allow comparison of RNA loaded.

 


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  		Fig. 2. Immunocytochemistry (ICC) showing high efficacy of recombinant adenovirus infection in SH-SY5Y and IGR-N-91 cells. Untreated or uninfected cells (control of non-infected cells); cells infected by adenovirus expressing full-length p73{alpha} (Ad-TAp73{alpha}) or {Delta}Np73{alpha} (Ad-{Delta}Np73{alpha}) at MOI of 15 for 48 hours; cells infected by empty vector (Mock). Nearly 100% of treated cells showed p73 expression in the nucleus. (A) ICC of SH-SY5Y cells; (B) ICC of IGR-N-91 cells; (C) western blot of 1 µg of total protein extracts from SH-SY5Y cells (left panel), IGR-N-91 cells (middle panel), and of 50 µg endogenous total protein extracts (right panel); ß-actin was used as a protein loading control.

 


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  		Fig. 4. Immunocytochemistry (ICC) showing strong nuclear staining in Ad-TAp73 SH-SY5Y infected cells. (A) SH-SY5Y cells; (B) IGRN-91 cells. Parental cells (Uninfected), or cells infected with full-length p73{alpha} (Ad-p73{alpha}), with {Delta}Np73a (Ad-{Delta}Np73{alpha}) or with empty vector (Mock). In contrast to SH-SY5Y cells (A), ICC in IGR-N-91 cells led to a diffuse and faint staining in whole cells (B), regardless of the expressed p73 isoform. `Cont' represents control of second antibody.

 


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  		Fig. 5. Cell cycle distribution showing differential patterns induced by TAp73{alpha} overexpression. (A) FACS. (B) Histogram representation of cell cycle population: Sub-G1 (black columns); G1 (white columns); S (hatched); G2/M (dots); data are means of three independent experiments ± s.d. (bars). The proportion of Ad-TAp73 SH-SY5Y neuroblasts (Ad-p73{alpha}) that underwent Sub-G1 phase increased considerably (60%) compared to Ad-{Delta}Np73{alpha} infected cells (10%) and to the non-infected cells and cells infected by empty vector (5% and 9%, respectively), indicating cells entering apoptosis. Ad-TAp73 IGR-N-91 cells exhibited a cell cycle distribution different from that of SH-SY5Y cells: more of the population underwent G1 phase (62% in treated versus 48% in control) leading to cell cycle arrest mediated by p21 activation as confirmed by western blot analysis shown in Fig. 3A.

 


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  		Fig. 6. Northern blots showing mRNA expression level of target genes in response to Ad-p73 SH-SY5Y and IGR-N-91 infected cells. (A) MDM2, BTG2, BAX and PUMA mRNA expression. It is noteworthy that in SH-SY5Y cells, BAX and PUMA mRNA expression is upregulated only in Ad-TAp73{alpha}, indicating again the rapid apoptosis observed in such cells (see also FACS in Fig. 5). BTG2, the p53 target gene, known to be involved in the regulation of G1/S transition of the cell cycle (Duriez et al., 2002Go) is strongly stimulated in Ad-TAp73{alpha} but also stimulated in Ad-{Delta}Np73{alpha} as compared to uninfected cells or cells infected by empty vector (Mock), or to Ad-TAp73 IGR-N-91 cells, Ad-{Delta}Np73, whereas MDM2 mRNA level expression is highly stimulated in Ad-TAp73 IGR-N-91-infected cells compared to Ad-{Delta}Np73- or control and empty vector-infected cells. Total RNA (10 µg) was prepared 48 hours after infection and submitted to northern blotting with appropriate cDNA probe; ethidium bromide staining of 28S and 18S rRNAs was used as an RNA loading control (A, lower panel). (B) Western blots showing protein expression of MDM2 and BAX. ß-Actin was used as protein loading control.

 

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