Upregulation of p27 cyclin-dependent kinase inhibitor and a C-terminus truncated form of p27 contributes to G1 phase arrest

Abstract

Potent anti-cancer compounds FR901464 and its methyl-ketal derivative spliceostatin A (SSA) inhibit cell cycle progression at G1 and G2/M phases. These compounds bind to the spliceosome and inhibit the splicing reaction. However, the molecular mechanism underlying G1 arrest after SSA treatment remains unknown. In this study, we found that ~90% of SSA-treated cells arrested at G1 phase after cell cycle synchronization. SSA treatment caused upregulation of the p27 cyclin-dependent kinase inhibitor both at mRNA and protein levels. In addition to p27, we observed expression of p27*, a C-terminal truncated form of p27 that is translated from CDKN1B (p27) pre-mRNA accumulated after splicing inhibition. Overexpression of p27 or p27* inhibited the exit from G1 phase after a double thymidine block. Conversely, knocking down of p27 by siRNA partially suppressed the G1 phase arrest caused by SSA treatment. There results suggest that G1 arrest in SSA-treated cells is caused, at least in part, by upregulation of p27 and p27*.

Figure 1. SSA treatment causes G1 arrest.

(a,b) Eight hours after release from a double thymidine block, synchronized HeLa S3 cells were treated with MeOH or 10 ng/ml or 30 ng/ml of SSA. The cells were harvested at the indicated time points and cell cycle progression was analyzed using cytometry (n = 3). (c) Representative histograms from (b).

Figure 2. SSA treatment upregulates p27 at mRNA and protein levels.

(a) Gene structure of CDKN1B and a schema of production of p27 and p27*. Cyan boxes, light yellow boxes, and horizontal lines represent coding sequences in exons, untranslated regions of exons and introns, respectively. (b) Eight hours after release from a double thymidine block, synchronized HeLa S3 cells were treated with MeOH or 10 ng/ml or 30 ng/ml of SSA. The cells were harvested at the indicated time points and expression levels of p27 and p27* were analyzed by immunoblotting. Protein level of α-tubulin was analyzed as an internal control. (c) Total RNAs were prepared from the cells harvested at the indicated time points in (b) and analyzed by qRT-PCR to check the relative expression level of exon 1 (CDKN1B (Ex1)), the spliced form (CDKN1B (Ex1-Ex2)), and the unspliced form (CDKN1B (Ex1-Int1)) of the CDKN1B gene. Error bars indicate s.d. (n = 3). (d) Total RNAs were prepared as in (c) and analyzed by RT-PCR using primers annealing to p27 exon 1 and exon 2 to detect both spliced and unspliced forms.

Figure 3. Overexpression of p27 and p27* inhibits cell cycle progression at G1 phase.

(a) HeLa S3 cells were transfected with pcDNA3.1 (Vector), pcDNA3.1-FLAG-p27, or pcDNA3.1-FLAG-p27*. The transfectants were treated with thymidine to synchronize the cell cycle. After release from the thymidine block, the cells were harvested at the indicated time points and cell cycle progression was analyzed using a cytometer (n = 4). (b) Replot of data in (a). Error bars indicate s.d. (n = 4). Statistical significance was investigated by the unpaired two–tailed t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 4. SSA-induced G1 arrest was suppressed by knockdown of p27.

(a) Eighteen hours after treatment with thymidine, synchronized HeLa S3 cells were washed with fresh medium and transfected with p27 or control siRNA. Eight hours after the transfection, the cells were treated with thymidine again for 16 h. Eight hours after release from the second thymidine block, the cells were treated with SSA or MeOH and harvested at the indicated time points. (b) Representative histograms of the samples analyzed by the cytometer. (c) Proportion of the cells in each phase (n = 3). (d) Proportion of G1 arrested cells (n = 3). Error bars indicate s.d. (n = 3). Statistical significance was investigated by the unpaired two-tailed t-test (*P < 0.05; **P < 0.01; ***P < 0.001).