Figure 4.

IDO1 expression and activity is suppressed by IDO1 promoter hypermethylation in ER-positive breast cancer cells. Treatment of ER-positive breast cancer cells with the demethylating agent 5-Aza (10 μM, 5 d) followed by stimulation with IFNγ (1000 U/mL, 48 h) significantly increased (A) IDO1 mRNA expression relative to GAPDH and (B) IDO1 protein expression (n = 3, per group). (C) Similarly, Kyn production of cells treated with 5-Aza for 5 d and subsequent IFNγ-stimulation for 48 h (n = 3) measured by HPLC was significantly increased compared with non-5-Aza treated controls (n = 3). 5-Aza treatment of the ER-negative HCC1954 cells neither influenced (D) IDO1 mRNA expression nor (E) IDO1 protein expression. (F) Activity of the unmethylated and the methylated IDO1 promoter was measured by luciferase reporter assay after stimulation with IFNγ (8 h; 20 U/mL for ZR-75–1 and HCC1954, 100 U/mL for BT-474 and MDA-MB-231), indicating that DNA methylation reduces IDO1 expression. Results are expressed as means, error bars indicate s.e.m. Statistical significance was determined by Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001.