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Expression of full-length p53 and its isoform Deltap53 in breast carcinomas in relation to mutation status and clinical parameters.

Baumbusch LO, Myhre S, Langerød A, Bergamaschi A, Geisler SB, Lønning PE, Deppert W, Dornreiter I, Børresen-Dale AL - Mol. Cancer (2006)

Bottom Line: Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter.Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors.Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, 0310 Oslo, Norway. lars.baumbusch@medisin.uio.no

ABSTRACT

Background: The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity.

Results: In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Deltap53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Deltap53 have been predicted. We confirmed the expression of Deltap53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Deltap53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53.

Conclusion: Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

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Schematic representation of the full-length p53 and the alternative splice form Δp53. (A) The mRNA structure of exons VI to X of the full-length p53 and the Δp53 isoform are shown. The removed sequence in Δp53 is located in parts of exon VII, in exon VIII, and in a fraction of exon IX. The alternative splice cassette junction sequence, represented twice in the full-length p53 and once in the alternative splice form, is indicated in red. (B) Structural organization of the full-length p53 and Δp53 and its functional domains. p53 protein domain classification and their locations along the protein according to Swiss-Prot/TrEMBL [12]. Subdomains of main structures are indicated with various colors. Red lines mark the part eliminated by the splicing process of Δp53 and covering aa 257 to 322 of the DNA-binding domain and the complete non-structured spacer region with bipartite nuclear localization signal.
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Figure 1: Schematic representation of the full-length p53 and the alternative splice form Δp53. (A) The mRNA structure of exons VI to X of the full-length p53 and the Δp53 isoform are shown. The removed sequence in Δp53 is located in parts of exon VII, in exon VIII, and in a fraction of exon IX. The alternative splice cassette junction sequence, represented twice in the full-length p53 and once in the alternative splice form, is indicated in red. (B) Structural organization of the full-length p53 and Δp53 and its functional domains. p53 protein domain classification and their locations along the protein according to Swiss-Prot/TrEMBL [12]. Subdomains of main structures are indicated with various colors. Red lines mark the part eliminated by the splicing process of Δp53 and covering aa 257 to 322 of the DNA-binding domain and the complete non-structured spacer region with bipartite nuclear localization signal.

Mentions: Δp53 is a novel alternative splice form that differs from the full-length p53 form by lacking parts of exon 7, complete elimination of exon 8 and partial removal of exon 9 [24]. The uncommon splice mechanism involves two 7 base pair long cassettes with an identical sequence in exon 7 and exon 9, of which one is retained in the isoform (Figure 1A; for sequence details see Additional file 1). We estimated the splice site plausibility by analyzing the cassette sequence for exonic splicing enhancer (ESE) motifs using the ESEfinder [36]. Exonic enhancers are potential binding sites for splicing factors of the highly conserved serine/arginine-rich (SR) protein family. The cassette motif gives a high score (3.5) for the splice factor SF2/ASF protein and has some similarity with known signal sequences for alternative splicing (see review [37]). According to Swiss-Prot/TrEMBL structural protein domain classification [12] full-length p53 consists of a transcription activation domain (aa 1–44), a DNA-binding domain (aa 102–292), an unstructured spacer containing a bipartite nuclear localization signal (aa 305–321; a bipartite nuclear localization signal domain is defined as two adjacent basic amino acids with a spacer region of any 10 residue and at least three basic residues (Arg or Lys) in the five positions following the spacer region [38]), a tetramerization domain (aa 325–356), and a C-terminal regulatory domain (aa 368–387) (Figure 1B). In Δp53, this domain organization is modified by the removal of 66 amino acid (residues 257 to 322), which mainly disturbs the DNA-binding domain, and eliminates the spacer with the bipartite nuclear localization signal. The DNA-binding domain (aa 102–292) is truncated, but the 53bp2 SH3 domain remains intact, while the spacer with the bipartite nuclear localization signal domain (aa 305–321) is entirely removed.


Expression of full-length p53 and its isoform Deltap53 in breast carcinomas in relation to mutation status and clinical parameters.

Baumbusch LO, Myhre S, Langerød A, Bergamaschi A, Geisler SB, Lønning PE, Deppert W, Dornreiter I, Børresen-Dale AL - Mol. Cancer (2006)

Schematic representation of the full-length p53 and the alternative splice form Δp53. (A) The mRNA structure of exons VI to X of the full-length p53 and the Δp53 isoform are shown. The removed sequence in Δp53 is located in parts of exon VII, in exon VIII, and in a fraction of exon IX. The alternative splice cassette junction sequence, represented twice in the full-length p53 and once in the alternative splice form, is indicated in red. (B) Structural organization of the full-length p53 and Δp53 and its functional domains. p53 protein domain classification and their locations along the protein according to Swiss-Prot/TrEMBL [12]. Subdomains of main structures are indicated with various colors. Red lines mark the part eliminated by the splicing process of Δp53 and covering aa 257 to 322 of the DNA-binding domain and the complete non-structured spacer region with bipartite nuclear localization signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1636663&req=5

Figure 1: Schematic representation of the full-length p53 and the alternative splice form Δp53. (A) The mRNA structure of exons VI to X of the full-length p53 and the Δp53 isoform are shown. The removed sequence in Δp53 is located in parts of exon VII, in exon VIII, and in a fraction of exon IX. The alternative splice cassette junction sequence, represented twice in the full-length p53 and once in the alternative splice form, is indicated in red. (B) Structural organization of the full-length p53 and Δp53 and its functional domains. p53 protein domain classification and their locations along the protein according to Swiss-Prot/TrEMBL [12]. Subdomains of main structures are indicated with various colors. Red lines mark the part eliminated by the splicing process of Δp53 and covering aa 257 to 322 of the DNA-binding domain and the complete non-structured spacer region with bipartite nuclear localization signal.
Mentions: Δp53 is a novel alternative splice form that differs from the full-length p53 form by lacking parts of exon 7, complete elimination of exon 8 and partial removal of exon 9 [24]. The uncommon splice mechanism involves two 7 base pair long cassettes with an identical sequence in exon 7 and exon 9, of which one is retained in the isoform (Figure 1A; for sequence details see Additional file 1). We estimated the splice site plausibility by analyzing the cassette sequence for exonic splicing enhancer (ESE) motifs using the ESEfinder [36]. Exonic enhancers are potential binding sites for splicing factors of the highly conserved serine/arginine-rich (SR) protein family. The cassette motif gives a high score (3.5) for the splice factor SF2/ASF protein and has some similarity with known signal sequences for alternative splicing (see review [37]). According to Swiss-Prot/TrEMBL structural protein domain classification [12] full-length p53 consists of a transcription activation domain (aa 1–44), a DNA-binding domain (aa 102–292), an unstructured spacer containing a bipartite nuclear localization signal (aa 305–321; a bipartite nuclear localization signal domain is defined as two adjacent basic amino acids with a spacer region of any 10 residue and at least three basic residues (Arg or Lys) in the five positions following the spacer region [38]), a tetramerization domain (aa 325–356), and a C-terminal regulatory domain (aa 368–387) (Figure 1B). In Δp53, this domain organization is modified by the removal of 66 amino acid (residues 257 to 322), which mainly disturbs the DNA-binding domain, and eliminates the spacer with the bipartite nuclear localization signal. The DNA-binding domain (aa 102–292) is truncated, but the 53bp2 SH3 domain remains intact, while the spacer with the bipartite nuclear localization signal domain (aa 305–321) is entirely removed.

Bottom Line: Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter.Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors.Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Center, 0310 Oslo, Norway. lars.baumbusch@medisin.uio.no

ABSTRACT

Background: The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity.

Results: In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Deltap53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Deltap53 have been predicted. We confirmed the expression of Deltap53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Deltap53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Deltap53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53.

Conclusion: Expression of p53 is accompanied by the functionally different isoform Deltap53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Deltap53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

Show MeSH
Related in: MedlinePlus