TIP-1 and Radioresistance of Malignant Gliomas Figure 7. TIP-1 interacts with LZAP and enhances the protein interaction between LZAP and ARF within the irradiated D54 glioma cells. A) Putative TIP-1 interacting proteins identified through yeast two-hybrid screening of a human fetal brain cDNA library. B) Schematic illustration of the constructs used for studies of the protein interaction between TIP-1 and LZAP. The specific interaction of TIP-1 and LZAP was validated with pull down assay (C) with recombinant GST-fused TIP-1 (WT) protein or a mutant (MUT) with a dysfunctional PDZ domain, as well as co- immunoprecipitation assay (D) by using D54 cells that were co-transfected with recombinant plasmids as indicated. E) Co-IP assays to show that the TIP-1 presence enhanced the LZAP interaction with ARF within the irradiated D54 cells, whereas depletion of TIP-1 reduced the complex formation between LZAP and ARF within the irradiation glioma cells. Shown are representative images of at least two independent experiments. doi:10.1371/journal.pone.0045402.g007 the presence of TIP-1 modulated the functionality of LZAP towards p53 polyubiquitination and degradation. Finally, in a mouse model with subcutaneous xenografts of human malignant glioma, it was demonstrateed that depleting TIP-1 within glioma cells inhibited the tumor regrowth after IR treatment. Data provided in this study support one conclusion that TIP-1 is a novel regulator in the radioresistance of malignant gliomas. The putative roles of TIP-1 in the radiation resistance were summarized in Fig. 10. Considering the high TIP-1 expression levels in the advanced human malignant gliomas, this study suggested that antagonizing TIP-1 might represent one novel approach for an improved radiotherapy of the advanced malignant gliomas. p53 plays a central role in determining the cell fate after genotoxic stress by mediating the DNA-damage-induced cell cycle arrest, senescence or apoptosis. p53 is a short-lived protein that is normally expressed at low level owing to its interaction with HDM2. HDM2 functions as an E3 ubiquitin ligase and promotes the proteasome-mediated p53 protein degradation [5]. DNA damage triggers p53 phosphorylation that blocks the interaction of p53 with MDM2 leading to the stabilization and activation of p53 PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45402