5-azaC reduces the stability of total RNA and RRM2 mRNA. (A-B) 5-azaC causes a dose- and time-dependent decline in the total RNA level in MV4-11 (A) and K562 cells (B). (C) 5-azaC shortens the half-life (t1/2) of RRM2 but not abl or GAPDH mRNA. MV4-11 cells were treated with 1μM actinomycin D to block the de novo transcription in the presence or absence of 5-azaC. mRNA levels of RRM2 and 2 internal controls, abl and GAPDH, were measured by quantitative RT-PCR at the indicated time points after actinomycin D treatment. The data are presented as a percentage of the mRNA level measured at time 0 (without actinomycin D). mRNA half-lives were calculated and are indicated in the plots. (D) Blockade of protein synthesis facilitates 5-azaC–induced destabilization of RRM2 mRNA. MV4-11 cells were pretreated with cycloheximide to block protein synthesis, followed by treatment with 5-azaC and actinomycin D. Normalized mRNA levels are presented as the percentage of actinomycin D only, and are shown as means ± SD from triplicate experiments. *P < .05 compared with actinomycin D only; #P < .05 compared with treatment of both 5-azaC and actinomycin D.
