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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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p53 and Δp53 standard curve by qRT-PCR. Standard curve plotting showing CO (concentration) in log scale versus Ct (Cycle threshold). The fluorescence signal of the reporter dye (FAM) subtracted by the baseline signal of the passive reference dye (ROX) results in a ratio defined as the normalized reporter signal ΔRn. ΔRn increases with accumulating PCR cycles until it reaches a plateau. Ct represents the fractional cycle number at which significant increase in Rn above a baseline signal of the passive reference dye (ROX) can be detected. Standard curve points are based on serially diluted cDNAs of a mixture of 10 human cancer cell lines in a 1,5 orders of linear dynamic range. All samples were performed in triplets. Red quadrates illustrate data for p53 standard curve; blue squares show data for Δp53 standard curve of the same template. The red line linear represents regression of the standard quantity and the CT value for Δp53 and green line stand for linear regression of the standard quantity and the CT value for p53. The comparative Ct value between the two standard curves is 1.40.
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Figure 4: p53 and Δp53 standard curve by qRT-PCR. Standard curve plotting showing CO (concentration) in log scale versus Ct (Cycle threshold). The fluorescence signal of the reporter dye (FAM) subtracted by the baseline signal of the passive reference dye (ROX) results in a ratio defined as the normalized reporter signal ΔRn. ΔRn increases with accumulating PCR cycles until it reaches a plateau. Ct represents the fractional cycle number at which significant increase in Rn above a baseline signal of the passive reference dye (ROX) can be detected. Standard curve points are based on serially diluted cDNAs of a mixture of 10 human cancer cell lines in a 1,5 orders of linear dynamic range. All samples were performed in triplets. Red quadrates illustrate data for p53 standard curve; blue squares show data for Δp53 standard curve of the same template. The red line linear represents regression of the standard quantity and the CT value for Δp53 and green line stand for linear regression of the standard quantity and the CT value for p53. The comparative Ct value between the two standard curves is 1.40.

Mentions: Using qRT-PCR, the expression level of both full-length p53 and Δp53 mRNA were determined. Both isoforms were present in the Universal Human Reference of 10 human cell lines mixture and in the human breast tumor samples. The p53 and Δp53 mRNA levels were determined by standard curve measurements in a 1.5 orders of linear dynamic dilution range performed on the same plate. Both splice forms showed similar slopes and accordingly have equivalent target efficiencies (Figure 4). Under the presupposition of equivalent efficiencies the Comparative Ct (Cycle threshold) ΔΔCt method can be selected to compare normalized expression levels of different samples relative to a calibrator sample.


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)

p53 and Δp53 standard curve by qRT-PCR. Standard curve plotting showing CO (concentration) in log scale versus Ct (Cycle threshold). The fluorescence signal of the reporter dye (FAM) subtracted by the baseline signal of the passive reference dye (ROX) results in a ratio defined as the normalized reporter signal ΔRn. ΔRn increases with accumulating PCR cycles until it reaches a plateau. Ct represents the fractional cycle number at which significant increase in Rn above a baseline signal of the passive reference dye (ROX) can be detected. Standard curve points are based on serially diluted cDNAs of a mixture of 10 human cancer cell lines in a 1,5 orders of linear dynamic range. All samples were performed in triplets. Red quadrates illustrate data for p53 standard curve; blue squares show data for Δp53 standard curve of the same template. The red line linear represents regression of the standard quantity and the CT value for Δp53 and green line stand for linear regression of the standard quantity and the CT value for p53. The comparative Ct value between the two standard curves is 1.40.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: p53 and Δp53 standard curve by qRT-PCR. Standard curve plotting showing CO (concentration) in log scale versus Ct (Cycle threshold). The fluorescence signal of the reporter dye (FAM) subtracted by the baseline signal of the passive reference dye (ROX) results in a ratio defined as the normalized reporter signal ΔRn. ΔRn increases with accumulating PCR cycles until it reaches a plateau. Ct represents the fractional cycle number at which significant increase in Rn above a baseline signal of the passive reference dye (ROX) can be detected. Standard curve points are based on serially diluted cDNAs of a mixture of 10 human cancer cell lines in a 1,5 orders of linear dynamic range. All samples were performed in triplets. Red quadrates illustrate data for p53 standard curve; blue squares show data for Δp53 standard curve of the same template. The red line linear represents regression of the standard quantity and the CT value for Δp53 and green line stand for linear regression of the standard quantity and the CT value for p53. The comparative Ct value between the two standard curves is 1.40.
Mentions: Using qRT-PCR, the expression level of both full-length p53 and Δp53 mRNA were determined. Both isoforms were present in the Universal Human Reference of 10 human cell lines mixture and in the human breast tumor samples. The p53 and Δp53 mRNA levels were determined by standard curve measurements in a 1.5 orders of linear dynamic dilution range performed on the same plate. Both splice forms showed similar slopes and accordingly have equivalent target efficiencies (Figure 4). Under the presupposition of equivalent efficiencies the Comparative Ct (Cycle threshold) ΔΔCt method can be selected to compare normalized expression levels of different samples relative to a calibrator sample.

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