| Relevance: | The stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element can be used to investigate whether anticancer drugs can induce ARE-driven gene expression.In these cells, luciferase activity is increased up to 50-fold following treatment with 50 µmol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells are increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with L-buthionine-S,R-sulfoximine (BSO) does not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells can be suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhances induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induces endogenous AKR1C (AKR1C refers to AKR1C1 andAKR1C2), a target gene of Nrf2. Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy. |
| Description: | The ARE-luciferase reporter plasmid was generated using the pGL3-promoter vector containing an SV40 promoter upstream of the firefly luciferase gene. They differ in the number of copies of ARE sequences that have been inserted, in head-to-tail orientation, through Nhe I & Xho I restrictionsites upstream of the promoter-luc+ transcriptional unit. A plasmid was made containing eight copies of the ARE (5’-GTGACAAAGCA-3’, with the minimal functional sequence underlined) present in both rat GSTA2 and mouse gsta1; called pGL-8xARE. A linker with the sequence of 5’-CCC-3’ and 5’-GGG-3’ on the opposite strand was placed between individual cis-elements. |
| Model Type: | Reporter |
| Species/Origin: | Human |
| Phenotype Keywords: | |
| Disease Keywords: | Drug Metabolism & Toxicity |
| Recommended Growth Conditions: | DMEM with glutamax supplemented with 10% fetal bovine serum and antibiotics. Do not culture beyond 15 passages after revival. |
| References: | Wang et al., 2006. Cancer Research. 66:10983-94. PMID: 17108137 Read more |
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