indicating that p300 regulates p21 promoter activity independent of p53. In vivo studies To test whether PTTG1-mediated senescence regulates tumor development and anti-neoplastic drug responses in vivo, we evaluated HCT116 WT and PTTG12/2 cells using subcutaneous xenografted tumors. As shown in Fig. 6a, volumes differed in WT and PTTG12/2 tumors, starting from day 7 (12376493 vs 3 5016232 mm , p,0.05). On day 19, the average volume of control vehicle-treated WT tumors was 25596797 mm3, doxoru- bicin treated WT tumor was 20996529 mm3 (18% inhibition compared to control vehicle -treated WT tumors), control vehicle -treated PTTG12/2 tumors was 14166585 mm3 and doxorubi- cin treated PTTG12/2 tumors was 3296263 mm3, indicating a 77% inhibition (p,0.001) when compared to control vehicle- treated PTTG12/2 tumors. Twenty one days after tumor cell implantation, mice were sacrificed, tumors harvested and weighed. PTTG12/2 tumors were smaller than WT (1.3260.39 vs 0.5260.18 g, p,0.001) (Fig. 6b), confirming results of the tumor volume measurements. In doxorubicin treated groups, WT tumor weight was reduced to 0.9960.29 (25% inhibition), PTTG12/2 tumor weight was reduced to 0.160.02 g (80% inhibition, p,0.001). Body weights of control and treated mice were not different (data not shown), suggesting limited serious drug toxic effects. Tumor tissues frozen in liquid nitrogen were sectioned and stained. As subcutaneous xenografted tumors exhibited internal necrosis (data not shown), only peripheral tumor tissue was used for subsequent analysis. In WT subcutaneous tumors, 4% tumor cells were senescent in control vehicle-treated, compared to 7% in doxorubicin-treated group. In cells derived from excised PTTG12/2 subcutaneous tumors, 36% exhibited enhanced b- gal staining after doxorubicin treatment, compared to 10% in the control vehicle treated group (Fig. 6c). The number of p21 positive cells was increased in doxorubicin-treated PTTG12/2 tumors (Fig. 6d). Western blotting of tumor tissue extracts showed enhanced p21 levels in PTTG12/2 xenografted tumors, confirm- ing the b-gal staining results (Fig. 6e). Discussion In this study, we demonstrate that HCT116 PTTG12/2 cells display enhanced sensitivity to both doxorubicin and TSA. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA, suggesting that the PTTG1 mediated response might be due to senescence. Reintroduction of PTTG1 into HCT116 PTTG12/2 cells reversed the enhanced sensitivity and features of cell senescence, suggesting that the PTTG1 effect is specific. We also evaluated PTTG1 functions in SW620 cells. Knock down PTTG1 enhanced drug-induced senescence, con- firming that PTTG1 functions to suppress drug-induced senes- cence. Senescence is a tumor suppressive program. It restrains cell division, and is induced by age-linked telomere shortening, oncogene activation, DNA damage, oxidative stress or chemo- therapy [42,43]. Several lines of evidence indicate that senescence contributes to cancer cell response to treatment. Senescence is important for in situ radio-sensitization of human prostate cancer cells [44], and mice bearing tumors developing drug- induced senescence have enhanced prognosis following chemo- therapy as compared to those harboring tumors with defective senescence mechanisms [45]. The clinical doses of doxorubicin PTTG1 and Drug-Induced Senescence (0.13 to 1.5 nmol/g) [46] were found to cause senescence in vitro [32], suggesting a role for senescence in regulating doxorubicin efficacy. In addition to senescence, other mechanisms have been proposed for PTTG1 regulation of drug sensitivity. PTTG1 interacts with p53 to inhibit cancer cell apoptosis [16], and with the Ku heterodimer to inhibit DNA damage repair [47]. Cells devoid of PTTG1 demonstrated impaired non-homologous DNA end-joining and therefore may regulate cell responses to DNA damage-inducing agents [48]. However, we did not detect DNA damage caused by doxorubicin or TSA when treated with indicated doses, which are in consistence with previously reported [35]. As previous research in our laboratory showed p21 plays a key role in regulating senescence [20,27,49], we focused our research on investigating roles of PTTG1-p21 in drug-induced senescence. Low doxorubicin concentrations (0.005–0.02 uM) induced histone acetylation and promoted p21 expression, likely responsible for enhanced cell senescence. In HCT116 PTTG12/2 cells, knock down of p21 levels reduced the number of senescent cells, suggesting that p21 is critical for the enhanced senescence observed in HCT116 PTTG12/2 cancer cells. The specificity of this pathway was confirmed using TSA, which also induces p21 and histone acetylation levels in treated cells. However, knock down of p21 does not completely rescue features of enhanced senescence, which could be either due to an incomplete knock down or activation of other senescence pathways. The latter is more likely because HCT116 colon cancer cells devoid of p21 are senescent when treated with anti-cancer drugs [50]. Interestingly, although p21 levels are higher in HCT116 PTTG12/2 cells when treated with low dose doxorubicin and TSA, the p21 promoter did not show enhanced activity under the same treatments, suggesting that the p21 promoter luciferase assay did not fully recapitulate the p21 transcription process. Our results suggest higher levels of p53, Sp1, p300, acetylated histone H3 and H4, and lower levels of HDAC1 on the p21 promoter in PTTG12/2 cells. Both p53 and Sp1 recruit HDAC1 and p300 to the promoter region [39]. PTTG1 binds p53 and Sp1 and modulates their functions [9,16]. The results here showed more abundant p53, Sp1 and p300 on the p21 promoter in HCT116 PTTG12/2 cells. Whether PTTG1 forms a transcription complex with these factors on the p21 promoter requires further investigation. PTTG1 promotes tumor angiogenesis [24], but mechanisms underlying PTTG1-mediated angiogenesis, cell death and drug responses in vivo are unclear. As we observed enhanced drug- induced senescence both in vitro and in vivo, we reasoned that senescence may be a major cell arrest pathway involved. Although clinical doses of doxorubicin cause senescence in vitro [32,46], the true effect of low doses of doxorubicin requires further in vivo study. In conclusion, these results demonstrate that PTTG1 regulates drug-induced senescence, providing new insights for the role of PTTG1 in determining cancer cell responses to anti-neoplastic treatments. Supporting Information Figure S1 HCT116 PTTG12/2 cells are more sensitive to doxorubicin (Dox). a) HCT116 WT and PTTG12/2 cells expressing an empty vector (WT+EV and PTTG12/2 +EV), and PTTG1 re-introduced PTTG12/2 (PTTG12/2 +PTTG1) cells were plated in 96-well plates and DNA synthesis assessed at different time points (time 0 represents 0 hours after cell plating); b) HCT116 WT and PTTG12/2 cells expressing an empty vector PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23754