Figure 1. TIP-1 expression levels correlate with the survival probability of patients with gliobmatoma multiforme. Patients with elevated TIP-1 expression levels (log2 tumor/normal ratio .1.5) are defined as ‘‘TIP-1 high’’ group, those with the ratio less than 0.5 are defined as ‘‘TIP-1 low’’ group. Kaplan-Meier survival probability curves were developed with MedCalc. Statistical analysis was conducted with Log Rank Test (p,0.05). doi:10.1371/journal.pone.0045402.g001 parental cells, TIP-1 knockdown with shRNA sensitized both of D54 and U87 cells to IR, but had no significant impact on T98G cells (Fig. 2B). A dose modification factor (DMF) of 1.54 +/2 0.10 and 1.23 +/2 0.07 were obtained at the 0.10 survival fraction for TIP-1 knockdown in D54 and U87 cells, respectively. TIP-1 ectopic expression had a dose-modification factor as of 0.71 +/2 0.12 in D54 cells. TIP-1 expression levels correlate with the apoptosis rate of IR-treated D54 cells Apoptosis was detected with western blot analysis of caspase-3 cleavage and flow cytometric profiling of PI- and Annexin V- stained cells. Semi-quantitative measurements were used to calculate fold changes of the caspase-3 activation before and after IR treatment, the fold changes were normalized to the respective vector control (counted as 1). Even though D54 cells are extremely resistant to IR-induced apoptosis, TIP-1 knockdown caused a prominent increase in the caspase-3 cleavage, whereas an ectopic expression of TIP-1 was associated with a reduced caspase-3 cleavage at 24 hours post a single dose of 5 Gy irradiation in those cells (Fig. 3A). Flow cytometric profiles of cell apoptosis demonstrated a similar trend. At 24 hours post a single dose of 10 Gy irradiation, few (,3.3%) of D54 cells with an ectopic expression of TIP-1 underwent apoptosis compared to around 7% cell apoptosis for the vector control, while TIP-1 knockdown caused around 8.4% apoptosis compared to less than 5% cell apoptosis for cells with a control shRNA (Fig. 3B). This trend was extended to 48 hours post the irradiation in D54 cells. TIP-1 facilitates DNA damage repair after IR treatment within glioma cells DNA damage were detected with c-H2AX foci staining and comet assays [29]. In the c-H2AX foci staining assays, only the cells with more than 5 foci were counted as positives (Fig. 4A). The quantitative analyses (Fig. 4B) showed that the majority (,85%) of D54 cells contained IR-induced c-H2AX foci at 30 minutes after 5 Gy irradiation. Different ratio of c-H2AX foci-positives in the cells with various levels of TIP-1 expression were observed at 4 hours and even more remarkable at 24 hours post the IR treatment. Compared to the vector control which had around 15.1% of c- TIP-1 and Radioresistance of Malignant Gliomas H2AX foci-positive cells at 24 hours post 5Gy IR, D54 cells with an ectopic expression of TIP-1 had less (around 6.99%) of c- H2AX foci-positive cells at the same time point after such IR treatment. However, D54 cells with TIP-1 knockdown had around 29.5% to 35.8% c-H2AX foci-positives compared to the vector control which had only around 20% c-H2AX foci-positives at 24 hours post 5Gy IR We also observed a remarkable portion of the cells with TIP-1 knockdown were stained as c-H2AX foci positive even without IR treatment (Figs. 4B), suggesting a potential role of TIP-1 in maintaining the genomic integrity within glioma cells. Such impact of the TIP-1 expression on IR-induced DNA damage was also observed in neutral comet assays, in which DNA double strand-breaks leads to a ‘‘tail-like’’ morphology of cell nucleus after electrophoresis (Fig. 4C). Cells with such tail were counted to quantify the DNA damage in the cell population, while tail length (Tail Moment) of individual cells was measured to demonstrate the severity of the DNA damage in individual cells [33]. Compared to D54 cells transfected with a vector control that had ,18% tail-positive cells at 10 minutes after a single dose of 5 Gy irradiation, D54 cells with an ectopic TIP-1 expression were only 10% tail-positive under same condition (Fig. 4D). Similarly but more remarkably, TIP-1 knockdown increased the tail-positive cells from ,18% (shRNA control) to 31-34% (Fig. 4D) at 10 minutes after 5 Gy irradiation. Consistent to the c-H2AX foci staining, tail moment analyses (Fig. 4E) indicated that the cells with TIP-1 depletion experienced more severe genome integrity, even without IR treatment. TIP-1 suppresses p53 activation in the irradiated glioma cells Western blot analyses were applied to determine the pathway mediators that sense, transduce, or execute the cellular responses to the IR-induced DNA damage. We detected overall protein levels of p53, the phosphorylation status of Chk1 (Ser317), Chk2 (Thr68) and Histone H-3 (Ser10) within the whole D54 cell lysates before and after IR treatment. At the indicated time points, modulating the TIP-1 protein level had a minor impact on the phosphorylation status of histone-3, Chk1 or Chk2 (Figs. 5A, B). Interestingly, we found that an ectoptic expression of TIP-1 inhibited the p53 protein accumulation after IR, while the TIP-1 knockdown was associated with an elevated p53 protein accumu- lation after irradiation (Figs. 5A–C). With the presence of a proteasome inhibitor MG132, western blot analysis of p53 ubiquitination status further revealed that an ectopic expression of TIP-1 promoted the polyubiquitination of p53 protein, while the TIP-1 depletion markedly reduced the p53 protein poly- ubiquitination within the irradiated D54 cells (Fig. 5D). These observations suggested that p53 might mediate the TIP-1- regulated susceptibility of glioma cells to IR. This hypothesis is also support by the results from clonogenic assays (Fig. 2B), in which manipulating TIP-1 expression had a minor impact on T98G cells which express a p53 mutant, but had dramatic influence on the radiosensitivity of D54 and U87 cells with wild- type p53 protein. p53 mediates the TIP-1-regulated cellular response to IR of glioma cells The putative roles of p53 and its ubiquitination in the TIP-1 regulated radioresistance of glioma cells were studied with D54 cells. A pool of validated small interference RNAs (siRNAs) were selected to silence p53 expression in D54 cells with different TIP-1 expression levels (Fig. 6A). Clonogenic assays indicated that p53 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45402