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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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Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. (A) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. (B) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers (Table 1) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).
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fig2: Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. (A) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. (B) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers (Table 1) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).

Mentions: To further map the p53 mRNA transcribed in these cells, series of RT–PCR tests were performed using the forward primer, F2 (exon 8 position 1008th according to GenBank accession no. K03199), matched with different reverse primers, R3 (at the junction of exon 8/9, nt position 1124), R4, R5 (in exon 9 at positions 1154 and 1184, respectively), and R6 (in exon 10, at position 1230). The sequences of these primers are given in Table 1 and the results are presented in Figure 2A. SK-N-AS cDNA gave an amplified fragment of the same size as SH-SY5Y cDNA with the three primer pairs, F2/R3, F2/R4 and F3/R5. However, in contrast to SH-SY5Y, no fragment was obtained with SK-N-AS cDNA using the F2/R6 primer pair, which suggests the absence of exon 10 in SK-N-AS mRNA.


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)

Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. (A) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. (B) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers (Table 1) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).
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Related In: Results  -  Collection

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

fig2: Detection of p53 mRNA abnormalities in SK-N-AS (AS) in comparison with SH-SY5Y (SH) cells. (A) Amplification of p53 cDNA using primers from exons 8 to 10 as indicated below each arrow; for precise position see Table 1 and GenBank K03199: F2 (forward primer in exon 8); R3 (reverse primer at the junction of exon 8/9); R4 (the first moiety of exon 9); R5 (exon 9, 30 nt downstream R4); R6 (beginning of exon 10). Note that no amplification was observed in SK-N-AS from exon 10 (last lane), in contrast to SH-SY5Y. (B) RT–PCR from SK-N-AS compared to SH-SY5Y cells. Specific primers (Table 1) were used to amplify the DBD, the p53β isoform (β) and the C-terminal domain (C-ter).
Mentions: To further map the p53 mRNA transcribed in these cells, series of RT–PCR tests were performed using the forward primer, F2 (exon 8 position 1008th according to GenBank accession no. K03199), matched with different reverse primers, R3 (at the junction of exon 8/9, nt position 1124), R4, R5 (in exon 9 at positions 1154 and 1184, respectively), and R6 (in exon 10, at position 1230). The sequences of these primers are given in Table 1 and the results are presented in Figure 2A. SK-N-AS cDNA gave an amplified fragment of the same size as SH-SY5Y cDNA with the three primer pairs, F2/R3, F2/R4 and F3/R5. However, in contrast to SH-SY5Y, no fragment was obtained with SK-N-AS cDNA using the F2/R6 primer pair, which suggests the absence of exon 10 in SK-N-AS mRNA.

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