PTTG1 and Drug-Induced Senescence Figure 2. HCT116 PTTG12/2 and SW620 PTTG1 knock down (PTTG1-kd) cells exhibit enhanced drug-induced senescence. a) HCT116 WT, PTTG12/2 and PTTG1 re-introduced PTTG12/2 (PTTG12/2 +PTTG1) cells were plated in 96-well plates for 24 hours and treated with control vehicle (C) or TSA for another 48 hours. DNA synthesis was assessed by measuring BrdU incorporation; b) HCT116 WT and PTTG12/2 cells were plated for 24 hours and treated with control vehicle (C) or TSA (0.005 uM) for another 48 hours, fixed, stained for b-gal activity and cell number counted; c) SW620 WT, PTTG1 knock down (PTTG1-kd) cells and PTTG1-transfected (PTTG1-tf) cells were plated in 96-well plates for 24 hours and treated with control vehicle (C) or doxorubicin (Dox) for another 48 hours, DNA synthesis was assessed by measuring BrdU incorporation; d) SW620 WT, PTTG1 knock down (PTTG1-kd) cells and PTTG1 transfected (PTTG1-tf) cells were plated in 24-well plates for 24 hours, treated with control vehicle (C) or doxorubicin (0.02 uM) for another 48 hours, b-gal staining performed and positive cells counted. Each experiment was repeated three times. Results are expressed as mean6SD, n = 3, *p,0.05, **p,0.001. doi:10.1371/journal.pone.0023754.g002 expressing PTTG1 reduces p21 levels in both HCT116 and SW620 cells (Fig 3 c). Sp1 and p53 regulate p21 expression and senescence in PTTG12/2 cells Sp1 and p53 regulate p21 transcription [37], while PTTG1 enhances in vitro Sp1 binding [9] and also inhibits p53 transactivating activity [16]. We therefore evaluated p53 and Sp1 protein levels and show that HCT116 PTTG12/2 cells contain similar levels of p53 and Sp1 compared to WT cells (Fig. 3d). After treated with 0.08, 0.313, 1.25, 5 uM of doxorubicin, Sp1 levels did not change. p53 was only induced in cells treated with 1.25 and 5 uM doxorubicin (Fig. 3d). Knock- down of p53 reduced p21 levels in HCT116 WT and PTTG12/2 cells, and the number of cells exhibiting senescence were reduced from 58% to 27% with TSA treatment as assessed by b–Gal staining (Fig. 3b). Knockdown of Sp1 had a similar effect, with cell senescence observed in 25% of cells. Sp1 and p53 protein levels did not differ after treatment with 0.005–0.02 uM doxorubicin or TSA (Fig. 3d), suggesting that p21 up-regulation is not associated with Sp1 or p53 level changes. We then evaluated the Sp1 or p53 binding on p21 promoter. p21 promoter contains several Sp1 and p53 binding sites (Fig. 4a). We designed 2 primer pairs spanning 22500,+50 bp of the p21 promoter (Fig. 4a), with primer pair 1 for p53 and primer pair 2 for Sp1 binding evaluation. The difference in p53 binding between doxorubicin-treated WT and PTTG12/2 cells was small (1.7 vs 2.4 fold, Fig. 4b). p53 binding was enhanced ,3.4 fold in PTTG12/2 cells when treated with TSA. Sp1 binding to the p21 promoter in WT but not PTTG12/2 cells was enhanced ,6.1 fold (p,0.001) by doxorubicin (Fig. 4c). TSA enhanced Sp1 binding to the p21 promoter by ,6.7 fold (p,0.001, Fig. 4c). As Sp1 binding motifs are essential for p21 promoter activity [38], we assessed the effects of deleting two p53 binding sites on the p21 promoter. After transfection of pGL-basic plasmid containing different p21 promoter fragments, those treated transfectants containing two intact p53 binding motifs demon- strated 2-fold higher activity in HCT116 PTTG12/2 than in WT cells (Fig. 4d). Promoter constructs devoid of p53 binding motifs showed similar activity in the two cell lines, suggesting that p53 is required to regulate pGL-p21 promoter activity in both WT and PTTG12/2 cells. However, when we treated WT and PTTG12/2 HCT116 cells with 0.005 uM doxorubicin or TSA, neither showed enhanced pGL-p21 luciferase activity (Fig. 4d), suggesting that chromatin changes may be involved. Histone acetylation on the p21 promoter As histone acetylation exposes chromatin to enable protein binding to gene promoters, anti-acetylated histone H3 and anti- acetylated histone H4 were used to co-immunoprecipitate chromatin, and immunoprecipitated DNAs were evaluated using primers targeting the p21 promoter. Both primer pairs detected abundant enriched p21 promoters in HCT116 PTTG12/2 cells when co-immunoprecipitated with acetylated histone H3 (Fig. 5a) or H4 antibody (Fig. 5b). Primer 1 demonstrated ,2-fold enhanced acetylated histone H3 in HCT116 PTTG12/2 cells. Doxorubicin treatment resulted in 4.3-fold increase in WT and 6.2-fold increase in HCT116 PTTG12/2 cells (p,0.05), while TSA treatment resulted in 6-fold increased acetylated histone H3 in WT, and ,10-fold increase in PTTG12/2 cells (p,0.05). Primer 2 demonstrated a similar pattern (data not shown). We also used a pair of negative primers (forward: ATGGTTGCCACTG- GGGATCT; reverse: TGCCAAAGCCTAGGGGAAGA) that flank genomic DNA between the GAPDH gene and the chro- mosome condensation-related SMC-associated protein (CNAP1) gene. As these negative primers did not detect differences between HCT116 WT and PTTG12/2 cells before and after drug treat- ment, it is likely that enhanced histone acetylation levels observed in PTTG12/2 cells are specific for these specific regions including the p21 promoter. Histone acetylation levels are controlled by histone acetylation transferase (HAT) and histone deacetylase (HDAC) [33]. To test for global changes in HAT/HDAC activities, we measured HAT and HDAC activity in vitro, and showed no difference in WT and PTTG12/2 cells (data not shown). HAT accumulation or HDAC decrease on these promoters might therefore lead to elevated histone acetylation. P300, an E1A associated protein with properties of a transcriptional adapter contains intrinsic histone acetyl-transferase activity [39] and interacts with p53 and sp1 to induce histone acetylation at corresponding regions [39,40]. We therefore surveyed p300 binding on the p21 promoter using primer pair 1 (Fig. 5c), and show that in PTTG12/2 cells p300 binding is 1.7-fold higher than in WT cells. After doxorubicin treatment, p300 binding on the p21 promoter was increased 2.5- and 6.6-fold in WT and PTTG12/2 cells respectively. Cells treated with TSA demonstrated a similar enhancement of p300 binding. As HDAC-1 decreases histone acetylation levels and acts as a co-repressor with p53 and sp1 [41], enhanced histone acetylation in the p21 promoter may be due to decreased HDAC- 1 binding. After doxorubicin treatment, HDAC-1 binding increased in PTTG12/2 cells (from 0.35 to 0.9 fold, Fig. 5d), likely indicating a compensation for enhanced histone acetylation in the p21 promoter. HDAC-1 binding to the p21 promoter was not altered in TSA-treated HCT116 PTTG12/2 cells. Similar changes were found by using primer pair 2 (data not shown). To assess the involvement of p300 in regulating p21 promoter activity, we knocked down p300 using siRNA and evaluated p21 promoter activity through a luciferase reporter assay. As shown in Fig. 5e, knock down of p300 reduced the 3kb p21 promoter activity by ,52% in PTTG12/2 HCT116 cells. To evaluate role of p53 in p300 mediated p21 regulation, we used the p21 promoter devoid of the p53 binding site (p21-luc Dp53). P300 knock down reduced p21-luc Dp53 activity by 51% (Fig. 5e), suggesting that regulatory effects of p300 on the p21 promoter may occur independently of p53. p21-luc and p21-luc Dp53 exhibited a similar activity in HCT116 p532/2 cells, further PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23754