TIP-1 and Radioresistance of Malignant Gliomas Figure 2. TIP-1 knockdown sensitizes glioma cells to ionizing radiation. A) Western blot analyses of TIP-1 expression in D54, U87 and T98G cells in which TIP-1 protein expression was genetically manipulated with recombinant plasmids or shRNA respectively. Two independent shRNA sequences were used for TIP-1 knockdown. B) Survival fractions of D54, U87 and T98G cells after single dose X-ray radiation treatment were determined with clonogenic assays. Vector control and parental cells were included in the assays. Shown are representative data from at least three independent experiments performed in triplicate. doi:10.1371/journal.pone.0045402.g002 knockdown attenuated the biological impact of TIP-1 expression on the radiosensitivity of D54 cells (Fig. 6B). Evaluations based upon dose modification factors or two-sided Student’s t-test of the data sets indicated that, after p53 knockdown, D54 cells demonstrated radiosensitivity independent of the TIP-1 expression levels. This data suggests that TIP-1 regulated radioresistance of glioma cells, at least in part, through p53. Furthermore, inhibition of HMD-2 with nutlin-3 partially eliminated the effects of TIP-1 expression on the p53 polyubiquitination and protein accumula- tion in the irradiated D54 cells (Fig. 6C), indicating that HDM-2 is involved in the TIP-1 regulated p53 inhibition in glioma cells. TIP-1 physically interacts with LZAP within glioma cells To determine the mechanism by which TIP-1 modulates the p53 protein ubiquitination, a yeast-two-hybrid screening was conducted to identify TIP-1-interacting proteins. Among the hits from the screening (Fig. 7A), we have particularly focused on LZAP which contains a typical PDZ-binding motif at its C- terminal end (Fig. 7B) and had been characterized as a tumor suppressor that regulates tumor cell response to multiple genotoxic stresses including IR through binding ARF and modulating the HMD-2 mediated p53 ubiquitination and activation [12,13,34]. To validate the protein interaction between TIP-1 and LZAP, in vitro pull-down assays were conducted with recombinant GST- tagged TIP-1 proteins that were purified to homogeneity [25]. As shown in Figure 7C, the endogenous LZAP protein was only precipitated with the wild type (WT) of TIP-1 from D54 cell lysates. Neither GST control nor one TIP-1 mutant (MUT) that contains a dysfunctional PDZ domain (Fig. 7B) bound LZAP protein in the pull-down assay. To study the in vivo protein interaction, a LZAP mutant was created by replacing critical amino acids in the putative C-terminal PDZ-binding motif with alanine (Fig. 7B, from –GTSL to -GASA). The mutation was verified with DNA sequencing. The FLAG-tagged LZAP, wild PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45402