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

First published online 1 March 2005
doi: 10.1242/jcs.01704

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 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 Goldschneider, D. Articles by Douc-Rasy, S. Search for Related Content PubMed PubMed Citation Articles by Goldschneider, D. Articles by Douc-Rasy, S. Social Bookmarking                         What's this?

The neurogene BTG2^TIS21/PC3 is transactivated by Np73 via p53 specifically in neuroblastoma cells

David Goldschneider¹, Karine Million¹, Anne Meiller³, Hedi Haddada¹, Alain Puisieux², Jean Bénard¹, Evelyne May^3,* and Sétha Douc-Rasy^1,

¹ CNRS UMR 8126, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France
² INSERM U590, Centre Leon Bérard, 28 rue Laënnec, 69008 Lyon, France
³ CEA-CNRS UMR217, PB6, 92265 Fontenay-aux-Roses, France

View larger version (32K): [in a new window]   Fig. 1. BTG2TIS21/PC3 promoter activation by TA- and N-p73 in wt-p53 neuroblastoma cell lines but not in wt-p53 breast cancer line MCF-7. (A) Schematic representation of pGL3-reporter constructs containing p53 binding site (BS) at –119, as previously described (Duriez et al., 2002); long BTG2TIS21/PC3 promoter sequence fragments of 2700 bp (–2700) and short promoter fragment (–266). (B) SH-SY5Y, (C) IMR-32, (D) LAN-1 and (E) MCF-7 cells were transiently co-transfected with pBTG2-Luc plasmid carrying long (2700) or short (266) promoter sequences in the presence of either TA- or Np73 expressing vectors or empty control plasmids. Transfection techniques and luciferase activity measurements were carried out as described in the Materials and Methods. Error bars represent standard deviation calculated from three independent experiments. Cells were transfected with empty vector, TAp73, Np73, p53DD, TAp73 + p53, Np73 + p53 as indicated.

View larger version (17K): [in a new window]   Fig. 2. Western blot showing p73, p63 and p53 endogenous protein expression in SH-SY5Y, IMR-32, LAN-1 and MCF-7 cell lines. Total protein extracts were obtained as previously described (Goldschneider et al., 2004) and 30 µg total lysate was loaded for western blotting. The blot on the right was overexposed for 30 minutes rather than 0.5 minutes as for the blots on the left.

View larger version (14K): [in a new window]   Fig. 3. Transcriptional activity of Waf1/p21 p53BS is inhibited by Np73. The mean relative luciferase activity±s.e.m. is shown for wt-p53 SH-SY5Y (A) and MCF-7 (B) cells co-transfected with 0.5 µg pE1-hWAF1 and 1 µg of plasmid encoding either Np73 or Np73ß. The p53DD effect is also shown.

View larger version (21K): [in a new window]   Fig. 4. Effect of ectopic expression of either TA- or Np73 on the endogenous expression of Waf1/p21 and BTG2TIS21/PC3 in wt-p53-expressing SH-SY5Y and MCF-7 cell lines. The transcript levels of Waf1/p21 (A,C) and BTG2TIS21/PC3 (B,D) were estimated by SYBR®-Green RTQ-PCR from total RNA extracted from SH-SY5Y (A,B) or MCF-7 (C,D) cells infected with either TA- or Np73-recombinant adenovirus. Results are mean expression levels±s.e.m. compared with those obtained from either uninfected cells or cells infected with an empty vector

View larger version (20K): [in a new window]   Fig. 5. BTG2 transactivation is not induced by p73ß isoforms. Transcriptional activity of BTG2TIS21/PC3 was analyzed by luciferase assays in wt-p53 SH-SY5Y (A), IMR-32 (B) and MCF-7 (C) cells co-transfected with 0.5 µg of luciferase reporter plasmid (BTG2 2700 or BTG2 266) and 1 µg of either Np73ß or p53DD expression plasmids. The values represent mean relative luciferase activity±s.e.m.

View larger version (25K): [in a new window]   Fig. 6. No accumulation of BTG2 endogenous mRNA in Ad-p73ß-infected SH-SY5Y cells. Comparative RTQ-PCR analyses of BTG2TIS21/PC3 mRNA levels in SH-SY5Y (A) and MCF-7 cells (B) infected with either p73 or p73ß isoforms.

View larger version (43K): [in a new window]   Fig. 7. Western blot showing an accumulation of p53 protein in response to an ectopic expression of either the or ß isoforms of TA- and Np73 proteins.

View larger version (14K): [in a new window]   Fig. 8. Schematic representation of the p53BS of three p53-target genes showing the pentamer orientation in each decamer. (a) Head-to-head (HH) for the canonical 20-bp p53-binding site, as in the Waf1/p21 promoter; (b) in tandem, head-to-tail (HT), as in the MDR-1 promoter; and (c) tail-to-tail (TT) as in the BTG2TIS21/PC3 promoter. Pu, purine; Py, pyrimidine.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter