Fig. 3.
MTA's effect on cFLIP mRNA expression in colon cancer cells requires de novo RNA synthesis but not de novo protein synthesis. A and B, RKO cells were pretreated with either 5 μg/ml actinomycin D (Act D) or vehicle (EtOH) for 1 h before either 1 mM MTA treatment or vehicle control (dimethyl sulfoxide) for 1 to 4 h. RNA was extracted at each time point, and cFLIP expression was assayed by quantitative real-time PCR analysis for cFLIPL (A) and for cFLIPS (B). Results are expressed as the average ± S.E. of five independent experiments. Linear regression analysis with bestfit lines provided formulas for the calculation of t½. *, p < 0.01 versus Act D + MTA. C and D, de novo protein synthesis is not required for cFLIP mRNA down-regulation by MTA in colon cancer cells. RKO cells were pretreated with either 10 μg/ml cycloheximide (CHX) or vehicle (EtOH) for 1 h before treatment with either 1 mM MTA or vehicle control (dimethyl sulfoxide) for 1 to 4 h. RNA was extracted for each time point, and cFLIP expression levels were determined by quantitative real-time PCR for cFLIPL (C) and for cFLIPS (D). Results are expressed as the average ± S.E. of three to five experiments.