TIP-1 and Radioresistance of Malignant Gliomas Figure 6. p53 mediates the TIP-1-regulated cellular response to IR of glioma cells. A) Western blot analysis of p53 expression within D54 cells with variable TIP-1 expression levels that were transfected with a control or p53-targeted siRNAs, respectively. Cell lystates were analyzed 72 hours post the siRNA transfection. B) Cell survival fractions in clonogenic assays after radiation treatment at variable doses (0, 2, 4, 6, and 8 Gy). Shown are the representative data of at least three independent experiments performed in triplicate. Significance was determined by comparing the denoted group to the respective vector control. * p,0.05, ** p.0.05, all tests of significance were determined with a two-sided Student’s t test. C) p53 ubiquitination status with or without HDM2 inhibitor (Nutlin-3). D54 cells with or without TIP-1 ectopic expression were incubated with DMSO (vehicle control) or Nutlin-3 (10 mM in DMSO) for 2 hours before the culture was mock treated or with IR (5 Gy). MG132 was added at 8 hours post the IR treatment and incubated for 4 more hours before the cell lysates were collected to analyze the p53 ubiquitination as described. The representative image shows the ubquitinated p53 ladder (pointed with arrows), as well as overall p53, TIP-1 and actin in each cell lysate. doi:10.1371/journal.pone.0045402.g006 in FoxN1-null nude mice. It was found that TIP-1 depletion retarded the tumor growth in nude mice (Fig. 9 and unpublished data). To determine the impact of the TIP-1 expression status on the radiation response of tumor in vivo, fractions of X-ray radiation (3 Gy for five consecutive days) was delivered when the tumors reached same size (around 3 mm in diameter). In the treatment course, only the tumors were irradiated while the other parts of mouse body were shielded. Tumor size was measured with caliper in and beyond the treatment courses. Compared to the tumors with a control shRNA that regrew after the treatment course (Fig. 9), the D54 xenografts with a TIP-1-specific shRNA did not assume tumor growth after the IR treatment course. Indeed, tumors eventually shrunk and some were undetectable by day 30 post the IR treatment. Discussion The majority of cancer patients have received radiotherapy in their tour to fight against cancers. Unfortunately, the therapeutic efficacy of radiation is largely limited by intrinsic or acquired resistance in patients with advanced malignant gliomas (grade III or IV). A significant portion of cancer research is to identify the genetic or epigenetic factors that contribute to the radioresistance and eventually could be targeted to improve the therapeutic efficacy of radiotherapy of cancer. This study has identified TIP-1 as one novel protein that confers the radioresistance in malignant glioma cells. Studies with established glioma cell lines demonstrat- ed that expression of TIP-1 conferred resistance of glioma cells to X-ray radiation while TIP-1 knockdown sensitized glioma cells to IR. Biochemical studies further identified that the TIP-1 regulates the radioresistance of glioma cells, at least in part, through promoting p53 protein polyubiquitination for degradation. LZAP was identified as one novel TIP-1-interacting protein in this study, PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45402