Limits...
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.

Show MeSH

Related in: MedlinePlus

Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 (*), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1636465&req=5

fig9: Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 (*), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.

Mentions: An investigation into the p53 genomic status and functions of eight human NB lines revealed that all five of the mutated cell lines had distinct genetic characteristics as is schematically represented in Figure 9: SK-N-BE(2) with a single missense mutation in the p53 gene, encoding a highly stable full-length protein. SK-N-AS and IGR-NB8 proteins, although they have intact transactivation and DBDs, were truncated at the C-terminus generating 341 and 326 amino acids respectively; they therefore lack the tetramerization domain that is essential for an active conformation. Very recently, Bourdon et al. (22) reported the putative occurrence of β and γ isoforms from different tissues due to alternate splicing that indicates the similarity to those of p73 and p63 as identified previously by Daniel Caput and co-workers (29). In the p53 isoforms scheme proposed by Bourdon et al. (22), the SK-N-AS cell line that elicits p53i9 protein expression is consistent with the p53β isoform. Genomic analysis reveals that the only occurrence of the p53β isoform in SK-N-AS results from a deletion spanning the intron 9/exon 10 junction. Similar to the p53β isoform in SK-N-AS, the p53 in IGR-NB8 that lacks 67 amino acids at C-terminus was, alone, unable to induce p21/WAF1 promoter activation except with endogenous wt-p53 on SH-SY5Y cells where transfection with IGR-NB8 significantly augmented the transcriptional activation of the p21/Waf-1 promoter (Figure 5A). Studies by other authors have reported the interaction between the C-terminal domain and another region that impedes the active conformation of p53, suggesting an allosteric model for p53 activity regulation (30). Such events have been demonstrated for the 342-stop mutant, generated by mutagenesis, which can modulate transactivation, growth and apoptosis (31). Moreover, Harms and Chen (32) reported that the C-terminal basic domain inhibits induction of the proapoptotic target gene insulin-like growth factor binding protein 3, suggesting that IGR-NB8 might induce this gene. IGR-N-91 had an abnormally high molecular weight protein due to the duplication of wild-type exons 7-8-9, thus affecting the DBD and OD; and LAN-1, with a mutation at codon 182 (Cys→stop) concurred with an earlier report showing extremely low levels of mRNA and undetectable protein expression (9).


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)

Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 (*), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Structure of p53 proteins in different neuroblastoma cell lines. The three functional domains are represented: TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain. The wild-type p53 gene in SH-SY5Y, IMR-32 and LAN-5 cells contains 11 exons that encode 393 amino acids. In SK-N-BE(2) cells, p53 is mutated at codon 135 (*), which converts cysteine to phenylalanine. In IGR-N-91 cells, a duplication of exons 7-8-9 adds an additional 107 amino acids leading to a total of 500. In SK-N-AS cells, a mutation due to alternate splicing downstream of exon 9 leads to a protein of 341 amino acids whereas in IGR-NB8 cells, the p53 protein ends at 326 amino acids owing to the mutation E326STOP.
Mentions: An investigation into the p53 genomic status and functions of eight human NB lines revealed that all five of the mutated cell lines had distinct genetic characteristics as is schematically represented in Figure 9: SK-N-BE(2) with a single missense mutation in the p53 gene, encoding a highly stable full-length protein. SK-N-AS and IGR-NB8 proteins, although they have intact transactivation and DBDs, were truncated at the C-terminus generating 341 and 326 amino acids respectively; they therefore lack the tetramerization domain that is essential for an active conformation. Very recently, Bourdon et al. (22) reported the putative occurrence of β and γ isoforms from different tissues due to alternate splicing that indicates the similarity to those of p73 and p63 as identified previously by Daniel Caput and co-workers (29). In the p53 isoforms scheme proposed by Bourdon et al. (22), the SK-N-AS cell line that elicits p53i9 protein expression is consistent with the p53β isoform. Genomic analysis reveals that the only occurrence of the p53β isoform in SK-N-AS results from a deletion spanning the intron 9/exon 10 junction. Similar to the p53β isoform in SK-N-AS, the p53 in IGR-NB8 that lacks 67 amino acids at C-terminus was, alone, unable to induce p21/WAF1 promoter activation except with endogenous wt-p53 on SH-SY5Y cells where transfection with IGR-NB8 significantly augmented the transcriptional activation of the p21/Waf-1 promoter (Figure 5A). Studies by other authors have reported the interaction between the C-terminal domain and another region that impedes the active conformation of p53, suggesting an allosteric model for p53 activity regulation (30). Such events have been demonstrated for the 342-stop mutant, generated by mutagenesis, which can modulate transactivation, growth and apoptosis (31). Moreover, Harms and Chen (32) reported that the C-terminal basic domain inhibits induction of the proapoptotic target gene insulin-like growth factor binding protein 3, suggesting that IGR-NB8 might induce this gene. IGR-N-91 had an abnormally high molecular weight protein due to the duplication of wild-type exons 7-8-9, thus affecting the DBD and OD; and LAN-1, with a mutation at codon 182 (Cys→stop) concurred with an earlier report showing extremely low levels of mRNA and undetectable protein expression (9).

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