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Expression of C-terminal deleted p53 isoforms in neuroblastoma.

Goldschneider D, Horvilleur E, Plassa LF, Guillaud-Bataille M, Million K, Wittmer-Dupret E, Danglot G, de Thé H, Bénard J, May E, Douc-Rasy S - Nucleic Acids Res. (2006)

Bottom Line: Xirodimas, M.Saville and D.Lane (2005) Genes Dev., 19, 2122-2137].

View Article: PubMed Central - PubMed

Affiliation: Centre National de Recherche Scientifique, UMR 8126, Institut Gustave Roussy, 94805 Villejuif, France.

ABSTRACT
The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

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p53 transactivation ability using yeast-based assay (FASAY). (A) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). (B) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).
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fig4: p53 transactivation ability using yeast-based assay (FASAY). (A) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). (B) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).

Mentions: It is possible to detect p53 mutation using a simple yeast colony color assay as described by Flaman et al. (23). When the strain is transformed with a plasmid-encoding wt p53, the cells express the ADE2 gene and produce white colonies (Figure 4A, a, b2 and c1, and Figure 4B, dish a). Cells containing mutant p53 fail to express ADE2 and form small red colonies (Figure 4A, b and b1, and Figure 4B, dish c). When the p53 cDNA fragment is deleted, cells are unable to form a colony (Figure 4A, c and c2). As shown in Table 2, FASAY was performed as a p53-standard test with full-length cDNA or with the split version at the 5′ and 3′ end (15). The background of FASAY experiments is around 10%. p53 wt expressing SH-SY5Y and LAN-5, 2 wt cell lines, yielded ∼92–97% of white colonies (Table 2).


Expression of C-terminal deleted p53 isoforms in neuroblastoma.

Goldschneider D, Horvilleur E, Plassa LF, Guillaud-Bataille M, Million K, Wittmer-Dupret E, Danglot G, de Thé H, Bénard J, May E, Douc-Rasy S - Nucleic Acids Res. (2006)

p53 transactivation ability using yeast-based assay (FASAY). (A) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). (B) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636465&req=5

fig4: p53 transactivation ability using yeast-based assay (FASAY). (A) Schematic representation of the analysis of p53 mutants using the yeast homologous recombination expression vector pRDI-22 carrying the 5′ and 3′ ends of the p53 open reading frame and the split forms, pFW35 and pFW34 (lacking p53 fragment from amino acids 66 to 210 for split 5′ and 211–348 for split 3′, respectively) transfected into YPH500 Ade2 yeast strain. This strain repairs double-strand breaks in transfected plasmids by homologous recombination as ‘gap repair’ (see text). (B) Photographs of yeast colonies showing 100% wild-type p53 where all colonies are white (a), or special mutated p53 by duplication of exons 7–9 found in IGR-N-91 cells (b), where white and red colonies were mixed (see also Table 2), and mutated p53 such as those in SK-N-BE(2), where all colonies are red (c).
Mentions: It is possible to detect p53 mutation using a simple yeast colony color assay as described by Flaman et al. (23). When the strain is transformed with a plasmid-encoding wt p53, the cells express the ADE2 gene and produce white colonies (Figure 4A, a, b2 and c1, and Figure 4B, dish a). Cells containing mutant p53 fail to express ADE2 and form small red colonies (Figure 4A, b and b1, and Figure 4B, dish c). When the p53 cDNA fragment is deleted, cells are unable to form a colony (Figure 4A, c and c2). As shown in Table 2, FASAY was performed as a p53-standard test with full-length cDNA or with the split version at the 5′ and 3′ end (15). The background of FASAY experiments is around 10%. p53 wt expressing SH-SY5Y and LAN-5, 2 wt cell lines, yielded ∼92–97% of white colonies (Table 2).

Bottom Line: Xirodimas, M.Saville and D.Lane (2005) Genes Dev., 19, 2122-2137].

View Article: PubMed Central - PubMed

Affiliation: Centre National de Recherche Scientifique, UMR 8126, Institut Gustave Roussy, 94805 Villejuif, France.

ABSTRACT
The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

Show MeSH
Related in: MedlinePlus