Figure 10. Schemetic illustration of the putative roles of TIP-1 in the radioresistance of malignant gliomas. doi:10.1371/journal.pone.0045402.g010 [35]. Activation of p53 is also regulated by ARF which releases p53 from the HDM2-mediated ubiquitination, or sequesters the HDM2-p53 complex in the cellular nucleus and prevents p53 degradation [6,36]. The complexity of p53 regulation was further demonstrated with discovery of up- and down-stream modulators. For example, in a human osteosarcoma cell line (U2OS), LZAP formed a ternary protein complex with ARF and HMDM2, and reversed the inhibitory ability of ARF on the HDM2’s ubiquitin ligase activity towards p53 degradation. Indeed, LZAP co- operated with ARF in maintaining p53 stability and increasing the p53 transcriptional activity probably through promoting mono-ubiquitination and nuclear translocation of p53 protein [12]. Opposite to those observations, this study provided evidences showing that TIP-1 is a novel interacting protein of LZAP in glioma cells, the presence of TIP-1 enhanced the protein interaction between LZAP and ARF, promoted the polyubiqui- tination of p53 and resulted in a reduced p53 protein accumu- lation after irradiation. We did not detect a significant difference regarding the ternary complex formed with LZAP, ARF and HMD2 or the subcellular distribution of p53 at variable TIP-1 expression levels (data not shown). Those data might in part reflect the complexity of p53 regulation and influence of cellular context in different type of tumors, but also suggest a new mechanism of action (MOA) of LZAP in human malignant glioma, that is, complex formation between LZAP and ARF might modulate the functionality of HMD2 towards the p53 polyubiquitination and proteasome-mediated degradation. Regarding the heterogeneous roles of LZAP in cancer biology, it was noted that a controversial function of LZAP was recently reported in human hepatocellular carcinomas [14]. TIP-1 has demonstrated a variety of biological functions, such as modulating the cell proliferation, migration and polarization through selective interaction with b-catenin [17], Rhotekin [20], glutaminase L [37], potassium channel Kir 2.3 [38] and others [19,21,22], respectively. Although several studies have connected TIP-1 with cell transformation and oncogenesis [18,19,25,39], yet its biological functions and the associated mechanisms remain largely elusive. This study provided the first ever evidences showing that TIP-1 is involved in the radioresistance of malignant gliomas. A time-course study showed that glioma cells with TIP-1 expression were more efficient to repair the IR-induced DNA damage, while TIP-1 knockdown delayed DNA repair in the irradiated glioma cells. The delayed DNA damage repair after IR was translated into an increased apoptosis and a reduced survival References 1. Norden AD, Drappatz J, Wen PY (2009) Antiangiogenic therapies for high- grade glioma. Nat Rev Neurol 5: 610–620. TIP-1 and Radioresistance of Malignant Gliomas fraction of the glioma cells with TIP-1 depletion. Interestingly, a significant amount of DNA damage were detected with comet assay and c-H2Ax foci staining (Fig. 4) in the TIP-1-depleted glioma cells even without IR treatment, suggesting that TIP-1 might plays a vital role in the genome stability or integrity of glioma cells. It was also noted that the impact of the TIP-1 expression status on apoptosis of the glioma cell was not as dramatic as that observed in clonogenic assays, suggesting more studies are needed to explore the putative roles of pathways other than apoptosis, such as senescence or auophagy [35], in the TIP-1 regulated glioma cell response to IR. Restoration of p53 functionality has been tested in preclinical and clinical settings as one approach to treat tumors. However, the outcomes are contrary due to the complexity of the p53 activation and its functionality in a diversified background of tumors [7,9,40]. Even though a significant portion of the primary malignant gliomas express the wild-type p53 protein, the status of p53 expression has a poor prognostic value in predicting the therapeutic outcomes of the primary malignant gliomas [10,11]. This study revealed a novel mechanism of p53 regulation in malignant glioma cells. In vitro studies indicated that the overexpressed TIP-1 in glioma cells inhibited p53 activation after irradiation, depleting TIP-1 resulted in an elevated accumulation of p53 protein and improved the cytotoxicity of IR. Studies with tumor xenografts showed that depleting TIP-1 inhibited the tumor regrowth after IR treatment, leading to tumor shrink or even disappearance in some animals. These data suggest that antago- nizing TIP-1 might represent one novel approach to improve the p53-targeted therapies of malignant gliomas. Supporting Information Figure S1 Impact of X-ray irradiation on the TIP-1 expression levels in D54 cells. (A) Monolayer of D54 cells were irradiated with variable doses of X-ray and cell lysates were prepared for SDS- PAGE and western blot profiling of protein expression at 12 hours post the irradiation. Mock (0 Gy) treated cells were used as control. (B) Monolayer of D54 cells were irradiated with 5 Gy of X-ray and cell lysates were prepared for SDS-PAGE and western blot profiling of protein expression at variable time points post the irradiation. Mock (0 Gy) treated cells were used as control. Actin was blotted in each sample. (PDF) Acknowledgments We thank Dr. Paul A. Welling at University of Maryland (Baltimore, MD) for providing TIP-1 constructs, and Dr. Wendell Yarbrough at Vanderbilt University for providing LZAP constructs that are essential for this work. We appreciate Ms. Halina Onishko for her technical contributions in the initial stage of this study. We are grateful to the excellent technical supports at the Flow Cytometry and Cell Imaging Cores of Vanderbilt University Medical Center. Author Contributions Conceived and designed the experiments: ZH. Performed the experiments: MH HW. Analyzed the data: MH HW ZH. Contributed reagents/ materials/analysis tools: HZ ZH. Wrote the paper: MH ZH. 2. Van Meir EG, Hadjipanayis CG, Norden AD, Shu HK, Wen PY, et al. (2010) Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma. CA Cancer J Clin 60: 166–193. PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e45402