High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated Tp53-responsive luciferase reporter.
Human Tp53 is normally a short-lived protein. Tp53 protein is stabilized and levels are increased in response to a variety of cellular stresses, including those induced by genotoxic anticancer drugs and environmental exposures. To engineer an efficient assay based on this property, we constructed and integrated a Tp53-specific reporter system into human cancer cells, termed p53R cells. We tested a range of conventional chemotherapeutic agents as well as over 16 000 diverse small compounds. Ionizing radiation and two-thirds of conventional chemotherapeutic agents, but only 0.2% of diverse compounds activated Tp53 activity by two-fold or greater, consistent with the presumptive genotoxic activation of Tp53 function. Cytotoxicity was independent of TP53 genetic status when paired, syngeneic wild-type TP53 and TP53-null cells in culture were treated with compounds that activated Tp53. From the unbiased survey of random compounds, Tp53 activation was strongly induced by an analog of AMSA, an investigational anti-cancer agent. Tp53 was also strongly induced by an N-oxide of quinoline and by dabequine, an experimental antimalarial evaluated in humans; dabequine was reported to be negative in other screens of mutagenicity and clastogenicity but carcinogenic in animal studies. Further exploration of antimalarial compounds identified the common medicinals chloroquine, quinacrine, and amodiaquine as Tp53-inducers. Flavonoids are known to have DNA topoisomerase activity, a Tp53-inducing activity that is confirmed in the assay. A reported clinical association of Tp53 immunopositive colorectal cancers with use of the antihypertensive agents was extended by the demonstration of hydralazine and nifedipine as Tp53-inducers. p53R cells represent an efficient Tp53 functional assay to identify chemicals and other agents with interesting biologic properties, including genotoxicity. This assay may have utility in the identification of novel chemotherapeutic agents, as an adjunct in the pharmaceutical optimization of lead compounds, in the exploration of environmental exposures, and in chemical probing of the Tp53 pathway.
Sohn
Taylor A
TA
Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, MD, USA.
Bansal
Ravi
R
Su
Gloria H
GH
Murphy
Kathleen M
KM
Kern
Scott E
SE
D000677:Amsacrine / Q000031:analogs & derivatives / Q000494:pharmacology; D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D016158:Genes, p53 / Q000187:drug effects*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010190:Pancreatic Neoplasms; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016588:Anticarcinogenic Agents; D000970:Antineoplastic Agents; D016159:Tumor Suppressor Protein p53; D000677:Amsacrine; D008156:Luciferases