PTTG1 and Drug-Induced Senescence activity and cells counted; d) HCT116 WT and PTTG12/2 cells were plated for 24 hours and treated with control vehicle (C) or different doses of doxorubicin for another 48 hours, Tunel assay performed and positive cells counted. Each experiment was repeated three times. Results expressed as mean6SD, n = 3, *p,0.05, **p,0.001. doi:10.1371/journal.pone.0023754.g001 Data analysis All results were expressed as mean6SD. Two group compar- ison was assessed by non-paired, two tail Student’s t-test. The difference between multiple groups was assessed by ANOVA. Results HCT116 PTTG12/2 cells exhibit enhanced drug-induced senescence We evaluated WT HCT116 and PTTG12/2 cell proliferation by measuring BrdU incorporation. As shown in Fig. 1a, with similar cell numbers plated, HCT116 WT and PTTG12/2 cells exhibited similar absorbance (OD 0.4) at 0 hour. After 48 hours, HCT116 WT cells showed an absorbance of 1.8 and PTTG12/2 cells 1.3 (28% decrease compared to WT, figure S1), suggesting that HCT116 PTTG12/2 cell number were less than WT. Reintroduction of PTTG1 into HCT116 PTTG12/2 cells (PTTG12/2 +PTTG1) increased cell number after 48 hours (see figure S2 for transfection efficiency), and these cells exhibited an OD value of 2.1 (1.6 fold increase compared to PTTG12/2 expressing an empty vector). We then evaluated doxorubicin- treated HCT116 WT and PTTG12/2 cells. As shown in Fig. 1b, doxorubicin (0.005 uM) inhibited BrdU incorporation by 8% in WT cells (OD value reduced from 1.8 in control vehicle group to 1.7 in doxorubicin group, not significant), and 42% in PTTG12/2 cells (OD value reduced from 1.3 in control vehicle group to 0.8 in doxorubicin group, p,0.001). Treatment with higher doses (0.02 uM) of doxorubicin inhibited BrdU incorporation by 52% in WT cells and 85% in PTTG12/2 cells (p,0.05). As lower doses (,0.1 uM) of doxorubicin cause senescence [32], and HCT116 PTTG12/2 cells were more sensitive to low dose doxorubicin as measured by BrdU incorporation, we reasoned that HCT116 PTTG12/2 cells may undergo enhanced drug-induced senes- cence. We therefore performed b-galactosidase (b–Gal) staining as a marker to evaluate cell senescence. As indicated in Fig. 1c, few cells stained positively when treated with control vehicle. After doxorubicin (0.005 uM) treatment, 56% of HCT116 PTTG12/2 cells exhibited enhanced b–Gal staining, compared to 5% of WT cells. We then tested whether apoptosis was involved in regulating HCT116 PTTG12/2cells response to doxorubicin. HCT116 WT and PTTG12/2 cells were Tunel assay positive only when treated with higher doxorubicin doses (1.25 uM, Fig. 1d). Cleaved caspase 3 was not detected in doxorubicin (0.005–0.02 uM) treated HCT116 WT cells even after 96 hours treatment (data not shown). Thus enhanced PTTG12/2 cell sensitivity to low doxorubicin doses is likely due to enhanced senescence rather than apoptosis. We detected ,51% increase of doxorubicin- induced senescence, but only ,30% decrease in cell numbers when treated with 0.02 uM doxorubicin. This result might be due to the fact that senescent cells are still metabolically viable. Over-expressing PTTG1 attenuates drug-induced senescence To assess whether enhanced senescence and cell sensitivity could be rescued, we reintroduced PTTG1 into HCT116 PTTG12/2 cells, and observed modest inhibition of BrdU incorporation as compared to HCT116 PTTG12/2 cells (42%, OD value reduced from 1.3 to 0.75) when treated with 0.005 uM doxorubicin (p,0.001) (Fig. 1b). b–Gal staining indicated fewer senescent cells when PTTG1 was re-introduced (6%) than in control HCT116 PTTG12/2 cells (56%, p,0.001, Fig. 1c). Trichostatin A (TSA) selectively inhibits histone deacetylase (HDAC) enzymes [33], interfering with removal of histone acetyl groups thereby altering transcription factor access to DNA. As TSA induces senescence in several cell lines without causing DNA damage [34,35], we tested whether HCT116 PTTG12/2 cells are more sensitive to TSA. As shown in Fig. 2a, treatment with 0.3 nM TSA inhibited BrdU incorporation by 52% (OD value reduced from 1.1 to 0.53) in HCT116 PTTG12/2 cells and 10% (OD value reduced from 1.5 to 1.35) in WT cells (p,0.001). b-gal staining showed that 75% of treated HCT116 PTTG12/2 cells were senescent compared to 5% of treated WT cells (p,0.001, Fig. 2b). When PTTG1 was reintroduced to PTTG12/2 cells, the BrdU incorporation inhibition by TSA was decreased to 6% (p,0.001 compared to 52% in PTTG12/2 cells, Fig. 2a). The induction of senescence was decreased to 6% (p,0.001 compared to 75% in PTTG12/2 cells, Fig. 2b), suggesting that PTTG1 attenuates the cell response to TSA treatment. We then tested whether PTTG1 attenuates drug responsiveness and drug-induced senescence in other cell types. SW620 colon cancer cells transfected with empty vector or PTTG1 plasmids or PTTG1 siRNA were treated with doxorubicin. In control vehicle treated cells, PTTG1 knock down cells (PTTG1-knock down) exhibited lower OD value (1.2) than WT (1.6, p,0.05 vs PTTG1- knock down) and PTTG1 transfected cells (1.6, p,0.05 vs PTTG1-knock down), suggesting that knock down of PTTG1 inhibited SW620 cell proliferation (Fig. 2c). After treatment with doxorubicin (0.005 uM), BrdU incorporation was inhibited by 62% (OD value reduced from 1.2 to 0.46, p,0.001), compared to 12% (OD value was reduced from 1.6 to 1.4) in WT cells. Senescence was enhanced in PTTG1-knock down cells (35%, p,0.05, Fig. 2d). SW620 cells transfected with PTTG1 exhibited inhibition of BrdU incorporation of 6% (OD value reduced from 1.8 to 1.69). In control (control vehicle-treated) group, few cells appeared senescent. These results indicate that PTTG1 attenuates SW620 colon cancer cell senescence and responsiveness to doxorubicin. p21 and drug-induced HCT116 PTTG12/2 cell senescence Doxorubicin-induced senescence is likely due to histone acetylation induced p21 elevation rather than DNA damage [35]. We evaluated the relative contribution of these two mechanisms to our observations. Consistent with previous reports [35], cells treated with low dose doxorubicin or TSA did not exhibit H2AX phosphorylation (data not shown), suggesting that DNA damage is not directly involved in drug-induced senescence. Chromatin acetylation facilitates access of transcriptional machin- ery to gene promoter regions [36], and we observed higher basal and treatment-induced acetylated histone H3 and H4 levels measured by western blot in HCT116 PTTG12/2 cells (Fig. 3a). To test the requirement of p21 for enhanced senescence observed in PTTG12/2 cells, we knocked down p21 and then assessed effects of cell treatments. PTTG12/2 HCT116 cells exhibited 21% senescent cells when p21 was suppressed, compared to 58% in control siRNA treated cells (p,0.001) (Fig 3 b), indicating the requirement for p21 in PTTG1 mediated senescence. More- over, HCT116 PTTG12/2 cells possess higher p21 levels. Over PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23754