| Abstrakt: |
Luciferase genes are widely used as reporters to analyze promoter and regulatory elements. We found that a luciferase reporter gene vector with a modified firefly luciferase gene (luc+), but not Renillaluciferase (Rluc), was induced by all-transretinoic acid (tRA) in the MCF-7 breast cancer cell line. tRA (5 × 10−6M) increased luciferase activity of the pGL3 promoter vector (containing luc+) up to ∼3.8-fold in MCF-7 cells, but not in LNCaP prostate cancer cells or JEG-3 choriocarcinoma cells. Chimeric plasmids were constructed and showed that tRA-induction required the luc+ gene, but not any specific promoter or vector sequence. Time course and dose-response studies of tRA-induction indicated that longer treatment (>24 h) and higher tRA dose (>10−6M) were required for luc+ induction compared with those for a positive retinoic acid response element (maximum induction at 6 h and 10−8M tRA). Studies with the translation inhibitor, cycloheximide, indicated the half-life of the luc+ protein was increased from 9.7 ± 1.5 to 22.1 ± 3.1 h with tRA treatment. Other retinoids, TTNPB, a retinoic acid receptor β/γ-specific ligand, and a retinoid X receptor ligand, did not significantly increase luc+ expression. Caution is needed in analysis of retinoid responsive gene regulation with the luciferase reporter system in MCF-7 cells, especially at high retinoid concentrations. |
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