A New p53 Cell-Based Assay
Summary Harvard researchers developed a reliable, high-throughput screening assay to identify activators and inhibitors of p53. This cell-based luminescence assay measures the level of a reporter gene product under the control of a promoter containing a p53-binding site.
P53 is a transcription factor that can activate multiple genes involved in cell cycle regulation, DNA repair and programmed cell-death. It is often described as the "gatekeeper" of cellular integrity and plays a central role in tumor suppression. It is the most frequently mutated protein in human cancers.
This assay features a triple read-out system where:
-Two cell lines express different reporter genes under the control of the same p53- binding promoter. -A third cell line expresses a third reporter gene under the control of a constitutive promoter.
Figure 1: Three reporter cell-lines in one assay well: the screen identifies compounds that specifically affect p-53-dependant firefly luciferase (FL) and secreted alkaline phosphatase (SEAP) expression without affecting CMV promoter-driven renilla luciferase (RL) expression.
The influence of flanking DNA-sequences is controlled by the first two cell-lines. General effects such as cytotoxicity are measured in the third cell-line. These build-in controls thus minimize non-specific effects to identify true hits in one screen.
In screening experiments, this assay has been performed on a 384-well plate format to screen on a diverse collection of 16,320 synthetic compounds and effectively selected several chemicals that specifically affect transcriptional activity of p53. We believe this assay can be successfully adapted to higher density plate formats such as 1536- and 6144- well plates.
Figure 2: Screen for chemicals which affect p53-dependant FL and SEAP expression without affecting CMV promoter-driven RL expression.
This inventive cell-based assay:
- Allows to screen compounds under physiological conditions;
- Identifies chemicals that specifically affect p53 expression;
- Controls non-specific effects;
- Eliminates the need for multiple screenings;
- Allows high-throughput screens.
Harvard's intellectual property portfolio includes the following patent: US 6,569,506 issued July 22, 2003.
Applications Cell-based assays identify cell-permeable molecules active within a physiological environment, in contrast to biochemical assays in which a compound is added to a purified or partially purified cell-extract. However, one major drawback of the currently available cell-based assay systems is the high false-positive rate resulting from non-specific effects within the cells. For Further Information Please Contact the Director of Business Development Katie Gordon Email: email@example.com Telephone: (617) 432-0920
Kim, Tae Kook
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