Presentation Title

Examination of Wild Type and Hotspot Mutant p53 Properties in vivo and in vitro

Faculty Mentor

Luiza Nogaj, Ph.D

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 44

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

The tumor suppressor and transcription factor p53 is a regulatory protein that participates in the prevention of cancer and plays a major role in cell processes such as apoptosis, DNA repair, and cell cycle. When mutated p53 is highly overexpressed in various types of cancer, it can no longer perform its function. In this study, the six hotspot mutations in the DNA binding domain were examined for their ability to bind to the mdm2 P2 promoter in vivo and in vitro. Their aggregation potential and effect on mammalian cell viability was also tested. Wild type and mutant p53 were transfected into mammalian cells and Chromatin Immunoprecipitation Assay (ChIP) was performed to determine the ability of p53 to bind to mdm2 P2 promoter in vivo. In this assay, protein-DNA complexes were formed and immunoprecipitated using p53 specific antibodies. The purified protein-DNA complexes were then quantified by a real time PCR. An immobilized template assay was then performed to assess the ability of p53 to bind to mdm2 P2 promoter in vitro. The mdm2-P2 promoter was amplified using a biotinylated mdm2-P2 primer. Biotinylated mdm2 P2 promoter DNA was incubated with WT and mutant p53 proteins and examined for their ability to bind to DNA. An OC antibody was used to test p53 aggregation in vitro. Both in vivo and in vitro results showed that some p53 mutants were able to bind to DNA (R248Q and R175C) while other mutants could not bind as well. All mutants excluding R175C were also aggregating. Further studies are necessary to determine the effects of the p53 mutants on cell proliferation.

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Nov 18th, 10:00 AM Nov 18th, 11:00 AM

Examination of Wild Type and Hotspot Mutant p53 Properties in vivo and in vitro

BSC-Ursa Minor 44

The tumor suppressor and transcription factor p53 is a regulatory protein that participates in the prevention of cancer and plays a major role in cell processes such as apoptosis, DNA repair, and cell cycle. When mutated p53 is highly overexpressed in various types of cancer, it can no longer perform its function. In this study, the six hotspot mutations in the DNA binding domain were examined for their ability to bind to the mdm2 P2 promoter in vivo and in vitro. Their aggregation potential and effect on mammalian cell viability was also tested. Wild type and mutant p53 were transfected into mammalian cells and Chromatin Immunoprecipitation Assay (ChIP) was performed to determine the ability of p53 to bind to mdm2 P2 promoter in vivo. In this assay, protein-DNA complexes were formed and immunoprecipitated using p53 specific antibodies. The purified protein-DNA complexes were then quantified by a real time PCR. An immobilized template assay was then performed to assess the ability of p53 to bind to mdm2 P2 promoter in vitro. The mdm2-P2 promoter was amplified using a biotinylated mdm2-P2 primer. Biotinylated mdm2 P2 promoter DNA was incubated with WT and mutant p53 proteins and examined for their ability to bind to DNA. An OC antibody was used to test p53 aggregation in vitro. Both in vivo and in vitro results showed that some p53 mutants were able to bind to DNA (R248Q and R175C) while other mutants could not bind as well. All mutants excluding R175C were also aggregating. Further studies are necessary to determine the effects of the p53 mutants on cell proliferation.