Improving Reporter Gene Assay Methodology for Evaluating the Ability of Compounds to Restore P53 Activity.

Tumor suppressor protein P53 induces cycle arrest and apoptosis by mediating the transcriptional expression of its target genes. Mutations causing conformational abnormalities and post-translational modifications that promote degradation are the main reasons for the loss of P53 function in tumor cells. Reporter gene assays that can scientifically reflect the biological function can help discover the mechanism and therapeutic strategies that restore P53 function. In the reporter gene system of this work, tetracycline-inducible expression of wild-type was used to provide a fully activated state as a 100% activity reference for the objective measurement of biological function. It was confirmed by RT-qPCR, cell viability assay, immunofluorescence, and Western blot analysis that the above-mentioned reporter gene system could correctly reflect the differences in biological activity between the wild-type and mutants. After that, the system was tentatively used for related mechanism research and compound activity evaluation. Through the tetracycline-induced co-expression of wild-type and mutant in exact proportion, it was observed that the response modes of typical transcriptional response elements (TREs) to dominant negative mutation effect were not exactly the same. Compared to the relative multiple-to-solvent control, the activity percentage relative to the 100% activity reference of wild-type can better reflect the actual influence of the so-called P53 mutant reactivator. Similarly, relative to the 100% activity reference, it can objectively reflect the biological effects caused by the inhibitor of negative factors, such as MDM2. In conclusion, this study provides a 100% activity reference and a reliable calculation model for relevant basic research and drug development.