Functional quantification of DNA-binding proteins p53 and estrogen receptor in cells and tumor tissues by DNA
Y Liu1, H Asch, M F Kulesz-Martin
1Department of Dermatology and Oregon Cancer Center, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Abstract:
Functional assays of proteins can monitor the consequences of defects attributable to posttranslational activating or inhibitory events as well as to genetic mutations. Such assays promise to permit evaluation of cooperating oncogenic or tumor suppressor pathways in cells and tumors. As a step toward realizing this promise, we designed the DNA affinity immunoblotting (DAI) method to measure the activities of multiple sequence-specific DNA-binding proteins simultaneously [initially p53 and estrogen receptor (ER)] in lysates of cells or frozen tumor tissues. DAI is a novel application of biotin/streptavidin affinity chromatography and immunoblotting. The p53 and ER proteins in cell or tissue lysates were bound to biotinylated, specific DNA probes, retrieved using a streptavidin-conjugated matrix, and then quantified in parallel with total protein by immunoblotting. The assay results were reproducible and specifically correlated with the known functional status of p53 in mouse and human cells of known p53 genotype, including those with low levels of p53 protein. ER immunohistochemistry of human breast samples, which is highly correlated with functional status and prognosis in human breast cancer, was also highly correlated with DNA binding activity results by DAI. In contrast, the p53 protein in cells is frequently expressed but inactive, potentially accounting for the lack of strict correlation of p53 immunohistochemical or mutational status with tumor response to chemotherapy. DAI offers a new means of molecular profiling and monitoring of p53 and other DNA-binding protein activities in cells and tumors. DAI has applications in the detection and identification of covalently modified forms of DNA-binding proteins and in the identification of their interacting proteins in complex with DNA.
Insights
We developed DNA affinity immunoblotting (DAI) to measure DNA-binding protein activity, offering a new way to profile protein function in cells and tumors. This method accurately assesses p53 and estrogen receptor (ER) activity, crucial for cancer research.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Functional protein assays are vital for understanding post-translational modifications and genetic mutations.
- Evaluating cooperating oncogenic or tumor suppressor pathways requires robust cellular and tumor analysis methods.
- Existing methods for assessing DNA-binding protein activity can be limited in scope and application.
Purpose of the Study:
- To design and validate a novel method, DNA affinity immunoblotting (DAI), for simultaneously measuring the activities of multiple sequence-specific DNA-binding proteins.
- To assess the utility of DAI for profiling p53 and estrogen receptor (ER) activity in cell and tumor lysates.
- To establish DAI as a reproducible and accurate tool for molecular profiling in cancer research.
Main Methods:
- Developed DAI, a method combining biotin/streptavidin affinity chromatography with immunoblotting.
- Utilized biotinylated DNA probes specific to target proteins (p53, ER) for capture from cell or tissue lysates.
- Quantified captured proteins using immunoblotting in parallel with total protein levels.
Main Results:
- DAI demonstrated reproducible results and specifically correlated with the known functional status of p53 across different cell types.
- Assay results for ER DNA-binding activity showed high correlation with established ER immunohistochemistry in human breast samples.
- DAI effectively measured protein activity even in samples with low protein levels or inactive protein forms.
Conclusions:
- DAI provides a novel and effective means for molecular profiling and monitoring of DNA-binding protein activities, including p53 and ER.
- The method offers insights into protein function that may not be apparent from immunohistochemical or mutational analysis alone.
- DAI has potential applications in detecting modified protein forms and identifying protein-DNA interactions in complex biological samples.
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