{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T21:42:42Z","timestamp":1780090962252,"version":"3.54.0"},"reference-count":46,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2022,11,10]],"date-time":"2022-11-10T00:00:00Z","timestamp":1668038400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Medical Research Fund of Guangdong Province","award":["A2021178"],"award-info":[{"award-number":["A2021178"]}]},{"name":"Medical Research Fund of Guangdong Province","award":["JCYJ20190813094603736"],"award-info":[{"award-number":["JCYJ20190813094603736"]}]},{"name":"Medical Research Fund of Guangdong Province","award":["JSGG20191129094408360"],"award-info":[{"award-number":["JSGG20191129094408360"]}]},{"name":"Medical Research Fund of Guangdong Province","award":["SZSM201612061"],"award-info":[{"award-number":["SZSM201612061"]}]},{"name":"Shenzhen Science and Technology Plan of Basic Research Projects","award":["A2021178"],"award-info":[{"award-number":["A2021178"]}]},{"name":"Shenzhen Science and Technology Plan of Basic Research Projects","award":["JCYJ20190813094603736"],"award-info":[{"award-number":["JCYJ20190813094603736"]}]},{"name":"Shenzhen Science and Technology Plan of Basic Research Projects","award":["JSGG20191129094408360"],"award-info":[{"award-number":["JSGG20191129094408360"]}]},{"name":"Shenzhen Science and Technology Plan of Basic Research Projects","award":["SZSM201612061"],"award-info":[{"award-number":["SZSM201612061"]}]},{"name":"Science, Technology and Innovation Committee of Shenzhen","award":["A2021178"],"award-info":[{"award-number":["A2021178"]}]},{"name":"Science, Technology and Innovation Committee of Shenzhen","award":["JCYJ20190813094603736"],"award-info":[{"award-number":["JCYJ20190813094603736"]}]},{"name":"Science, Technology and Innovation Committee of Shenzhen","award":["JSGG20191129094408360"],"award-info":[{"award-number":["JSGG20191129094408360"]}]},{"name":"Science, Technology and Innovation Committee of Shenzhen","award":["SZSM201612061"],"award-info":[{"award-number":["SZSM201612061"]}]},{"name":"Shenzhen\u2019s Sanming Project","award":["A2021178"],"award-info":[{"award-number":["A2021178"]}]},{"name":"Shenzhen\u2019s Sanming Project","award":["JCYJ20190813094603736"],"award-info":[{"award-number":["JCYJ20190813094603736"]}]},{"name":"Shenzhen\u2019s Sanming Project","award":["JSGG20191129094408360"],"award-info":[{"award-number":["JSGG20191129094408360"]}]},{"name":"Shenzhen\u2019s Sanming Project","award":["SZSM201612061"],"award-info":[{"award-number":["SZSM201612061"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 P53 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 P53 and mutant P53 in exact proportion, it was observed that the response modes of typical transcriptional response elements (TREs) to dominant negative P53 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 P53 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 P53 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.<\/jats:p>","DOI":"10.3390\/ijms232213867","type":"journal-article","created":{"date-parts":[[2022,11,10]],"date-time":"2022-11-10T19:16:23Z","timestamp":1668107783000},"page":"13867","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Improving Reporter Gene Assay Methodology for Evaluating the Ability of Compounds to Restore P53 Activity"],"prefix":"10.3390","volume":"23","author":[{"given":"Xinle","family":"Han","sequence":"first","affiliation":[{"name":"Biomedical Research Institute, Shenzhen Peking University\u2014The Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, China"},{"name":"Department of Pathology, Ninth People\u2019s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Du","sequence":"additional","affiliation":[{"name":"Department of Hematology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dandan","family":"Shi","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingjie","family":"Li","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dandan","family":"Li","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kun","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suwen","family":"Lin","sequence":"additional","affiliation":[{"name":"Biomedical Research Institute, Shenzhen Peking University\u2014The Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingzhong","family":"Zhu","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoufang","family":"Huang","sequence":"additional","affiliation":[{"name":"Ganzhou Key Laboratory of Hematology, Department of Hematology, The First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"You","family":"Zhou","sequence":"additional","affiliation":[{"name":"Shenzhen Chipscreen Biosciences Co., Ltd., Shenzhen Hi-Tech Industrial Park, Shenzhen 518057, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1182-6866","authenticated-orcid":false,"given":"Zhengyu","family":"Fang","sequence":"additional","affiliation":[{"name":"Biomedical Research Institute, Shenzhen Peking University\u2014The Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, China"},{"name":"Department of Pathology, Peking University Shenzhen Hospital, Shenzhen 518036, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1016\/0092-8674(95)90039-X","article-title":"Mice lacking p21CIP1\/WAF1 undergo normal development, but are defective in G1 checkpoint control","volume":"82","author":"Deng","year":"1995","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4131","DOI":"10.1182\/blood-2005-04-1595","article-title":"BH3-only proteins Puma and Bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo","volume":"106","author":"Erlacher","year":"2005","journal-title":"Blood"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1038\/cdd.2008.16","article-title":"In several cell types tumour suppressor p53 induces apoptosis largely via Puma but Noxa can contribute","volume":"15","author":"Michalak","year":"2008","journal-title":"Cell Death Differ."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1036","DOI":"10.1126\/science.1090072","article-title":"p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa","volume":"302","author":"Villunger","year":"2003","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"106013","DOI":"10.1016\/j.phrs.2021.106013","article-title":"p53: A double-edged sword in tumor ferroptosis","volume":"177","author":"Ji","year":"2021","journal-title":"Pharmacol. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1561","DOI":"10.1158\/1078-0432.CCR-11-3040","article-title":"The fundamental role of the p53 pathway in tumor metabolism and its implication in tumor therapy","volume":"18","author":"Shen","year":"2012","journal-title":"Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5961","DOI":"10.1038\/s41467-021-26213-y","article-title":"Cancer gene mutation frequencies for the U.S. population","volume":"12","author":"Mendiratta","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"a001008","DOI":"10.1101\/cshperspect.a001008","article-title":"TP53 mutations in human cancers: Origins, consequences, and clinical use","volume":"2","author":"Olivier","year":"2010","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1038\/nm.4333","article-title":"Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients","volume":"23","author":"Zehir","year":"2017","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1038\/nrc.2017.109","article-title":"Targeting mutant p53 for efficient cancer therapy","volume":"18","author":"Bykov","year":"2018","journal-title":"Nat. Rev. Cancer"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1038\/387296a0","article-title":"Mdm2 promotes the rapid degradation of p53","volume":"387","author":"Haupt","year":"1997","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.phrs.2015.03.006","article-title":"A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins","volume":"95\u201396","author":"Soares","year":"2015","journal-title":"Pharmacol. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"610820","DOI":"10.3389\/fonc.2020.610820","article-title":"TP53 Mutations in Acute Myeloid Leukemia: Still a Daunting Challenge?","volume":"10","author":"Molica","year":"2020","journal-title":"Front. Oncol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kogan, S., and Carpizo, D.R. (2018). Zinc Metallochaperones as Mutant p53 Reactivators: A New Paradigm in Cancer Therapeutics. Cancers, 10.","DOI":"10.3390\/cancers10060166"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1021\/acssensors.1c00002","article-title":"Single-Molecule FRET-Based Dynamic DNA Sensor","volume":"6","author":"Megalathan","year":"2021","journal-title":"ACS Sens."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1038\/nm0302-282","article-title":"Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound","volume":"8","author":"Bykov","year":"2002","journal-title":"Nat. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4749","DOI":"10.1158\/0008-5472.CAN-1305-2","article-title":"Ser392 phosphorylation regulates the oncogenic function of mutant p53","volume":"64","author":"Yap","year":"2004","journal-title":"Cancer Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"6442","DOI":"10.1038\/onc.2010.382","article-title":"PRIMA-1(MET)\/APR-246 targets mutant forms of p53 family members p63 and p73","volume":"29","author":"Shen","year":"2010","journal-title":"Oncogene"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1016\/0092-8674(91)90384-B","article-title":"Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation","volume":"65","author":"Milner","year":"1991","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1128","DOI":"10.1101\/gad.340976.120","article-title":"p53 tetramerization: At the center of the dominant-negative effect of mutant p53","volume":"34","author":"Lozano","year":"2020","journal-title":"Genes Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2330","DOI":"10.1038\/sj.onc.1207396","article-title":"Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes","volume":"23","author":"Willis","year":"2004","journal-title":"Oncogene"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.ejca.2017.06.023","article-title":"Mutant p53 as a target for cancer treatment","volume":"83","author":"Duffy","year":"2017","journal-title":"Eur. J. Cancer"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"41363","DOI":"10.18632\/oncotarget.9133","article-title":"COTI-2, a novel small molecule that is active against multiple human cancer cell lines in vitro and in vivo","volume":"7","author":"Salim","year":"2016","journal-title":"Oncotarget"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1126\/science.1183424","article-title":"Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML","volume":"328","author":"Zhang","year":"2010","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/s41698-021-00235-7","article-title":"Pharmacogenomics characterization of the MDM2 inhibitor MI-773 reveals candidate tumours and predictive biomarkers","volume":"5","author":"Vuaroqueaux","year":"2021","journal-title":"NPJ Precis. Oncol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3079","DOI":"10.1158\/0008-5472.CAN-20-0189","article-title":"Inhibition of MDM2 Promotes Antitumor Responses in p53 Wild-Type Cancer Cells through Their Interaction with the Immune and Stromal Microenvironment","volume":"81","author":"Wang","year":"2021","journal-title":"Cancer Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"491","DOI":"10.3389\/fphar.2016.00491","article-title":"P53-MDM2 Pathway: Evidences for A New Targeted Therapeutic Approach in B-Acute Lymphoblastic Leukemia","volume":"7","author":"Trino","year":"2016","journal-title":"Front. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1021\/ml4000657","article-title":"Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development","volume":"4","author":"Vu","year":"2013","journal-title":"ACS Med. Chem. Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3550","DOI":"10.1158\/1078-0432.CCR-15-1698","article-title":"Targeting MDM2 for Treatment of Adenoid Cystic Carcinoma","volume":"22","author":"Warner","year":"2016","journal-title":"Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3484","DOI":"10.1038\/sj.onc.1208419","article-title":"PRIMA-1(MET) synergizes with cisplatin to induce tumor cell apoptosis","volume":"24","author":"Bykov","year":"2005","journal-title":"Oncogene"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"E5271-80","DOI":"10.1073\/pnas.1610421113","article-title":"2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells","volume":"113","author":"Bauer","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1016\/j.ccr.2012.03.042","article-title":"Allele-specific p53 mutant reactivation","volume":"21","author":"Yu","year":"2012","journal-title":"Cancer Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.ccell.2020.11.013","article-title":"Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site","volume":"39","author":"Chen","year":"2021","journal-title":"Cancer Cell"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"543","DOI":"10.3390\/md13010543","article-title":"Activation of p53 with ilimaquinone and ethylsmenoquinone, marine sponge metabolites, induces apoptosis and autophagy in colon cancer cells","volume":"13","author":"Lee","year":"2015","journal-title":"Mar. Drugs"},{"key":"ref_35","first-page":"6021","article-title":"Microtubule-active drugs taxol, vinblastine, and nocodazole increase the levels of transcriptionally active p53","volume":"55","author":"Tishler","year":"1995","journal-title":"Cancer Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1038\/ncb1123","article-title":"Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex","volume":"6","author":"Leu","year":"2004","journal-title":"Nat. Cell Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7974","DOI":"10.1523\/JNEUROSCI.0897-06.2006","article-title":"Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats","volume":"26","author":"Endo","year":"2006","journal-title":"J. Neurosci. Off. J. Soc. Neurosci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/s13402-017-0361-5","article-title":"p53 expression and subcellular survivin localization improve the diagnosis and prognosis of patients with diffuse astrocytic tumors","volume":"41","author":"Faccion","year":"2018","journal-title":"Cell. Oncol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3250","DOI":"10.1074\/jbc.M805658200","article-title":"MSL2 promotes Mdm2-independent cytoplasmic localization of p53","volume":"284","author":"Kruse","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/j.tips.2013.11.005","article-title":"Regulation of transcription factor activity by interconnected post-translational modifications","volume":"35","author":"Filtz","year":"2014","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1016\/j.molcel.2006.05.015","article-title":"Structural basis of DNA recognition by p53 tetramers","volume":"22","author":"Kitayner","year":"2006","journal-title":"Mol. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"14327","DOI":"10.1073\/pnas.0907840106","article-title":"Ultraslow oligomerization equilibria of p53 and its implications","volume":"106","author":"Natan","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2682","DOI":"10.1002\/jcb.25592","article-title":"TP53 Mutations in Head and Neck Squamous Cell Carcinoma and Their Impact on Disease Progression and Treatment Response","volume":"117","author":"Zhou","year":"2016","journal-title":"J. Cell Biochem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.drup.2018.05.001","article-title":"How mutations shape p53 interactions with the genome to promote tumorigenesis and drug resistance","volume":"38","author":"Stiewe","year":"2018","journal-title":"Drug Resist. Updat."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1016\/S1097-2765(01)00213-1","article-title":"PUMA induces the rapid apoptosis of colorectal cancer cells","volume":"7","author":"Yu","year":"2001","journal-title":"Mol. Cell"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1589","DOI":"10.1093\/nar\/gkm1040","article-title":"Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA","volume":"36","author":"Veprintsev","year":"2008","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/22\/13867\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:14:02Z","timestamp":1760145242000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/22\/13867"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,10]]},"references-count":46,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2022,11]]}},"alternative-id":["ijms232213867"],"URL":"https:\/\/doi.org\/10.3390\/ijms232213867","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,10]]}}}