suppressor that physically associates with ARF and activates p53 in head-and-neck carcinoma cells [12]. It was found that LZAP regulates the susceptibility of breast cancer cells to genotoxic stresses including IR and several chemotherapeutic agents [13]. However, a recent report [14] has documented a controversial function of LZAP in hepatocellular carcinomas suggesting the complexity of its functionality under influence of cellular context in different type of tumors. TIP-1 was firstly identified through yeast two-hybrid screening as one protein interacting with HTLV-1’s tax protein [15]. After that, several more interacting partners and its biological functions in cell proliferation, stress response, development and cell polarization have been reported [16–22]. TIP-1 is conservative among species [18]. Compared to other PDZ proteins that usually contain multiple structural domains and thus function as scaffold proteins in the assembly of macromolecular machineries [23,24], TIP-1 protein is primarily composed of a single PDZ (PSD-95/ DlgA/ZO-1) domain. The unique structural identity suggests that TIP-1 might function differently from other PDZ proteins. TIP-1 demonstrated inhibitory activity on the beta-catenin-driven gene expression and proliferation of colon cancer cells [17]. It mediates the serum response through interacting with Rhotekin [20], it was identified as one essential components for the HPV-16 E6 oncoprotein-driven cell transformation [19]. We have character- ized the translocation of the predominantly intracellular TIP-1 protein onto the cell plasma membrane of tumor cells as one biomarker of tumor response to ionizing radiation (IR) before the onset of apoptosis or cell death [25]. Elevated expression of TIP-1 has been found in human invasive breast cancers and associated with pulmonary metastasis in mouse models [26]. However, the biological functions, particularly in oncology, of this unique PDZ domain protein are elusively unclear. In this report, we further studied the roles of TIP-1 in the IR response of malignant gliomas. Meta-analysis indicated that high levels of TIP-1 expression are associated with the poor prognosis of human malignant gliomas after radiotherapy. Studies with established cell lines in vitro and in vivo revealed a novel function of TIP-1 in the cellular response of human malignant gliomas to IR, and provided the first evidences suggesting that antagonizing TIP-1 might represent one novel approach to sensitize malignant gliomas to radiotherapy. Materials and Methods Cell culture, antibodies, and chemicals Human glioblatoma cell lines (D54, U87 and T98G) were obtained from Dr. Yancie Gillespie (University of Alabama- Birmingham, Birmingham, AL) and purchased from American Type Culture Collection (ATCC, Rockville, MD, USA), respec- tively. The cells were maintained in DMEM/F12 medium supplemented with 10% fetal calf serum, 1% sodium pyruvate, and 1% penicillin/streptomycin (Thermo Scientific Inc., Wal- tham, MA). Cells were irradiated with 300 kV X-rays using a Pantak Therapax DXT 300 X- ray unit (Pantak, East Haven, CT). Rabbit anti-human TIP-1 antibody was produced as described previously [25]. Antibodies against LZAP, HDM2, DNA-PKCS, p53, and Ub(P4D1) were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA). Antibodies recognizing the cleaved caspase-3, phospho-Chk1 (Ser317), phospho-Chk2 (Thr68), and phospho-Histone H3 (Ser10) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against actin and ARF, and HRP- or fluorescence-labeled secondary antibodies were obtained from Invitrogen (Carlsbad, CA). Nutlin-3 (one HDM-2 inhibitor) was purchased from Sigma and dissolved in DMSO as TIP-1 and Radioresistance of Malignant Gliomas stock. Other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Recombinant plasmids, mutagenesis, and protein production Recombinant pcDNA3.1 plasmids expressing cMyc-tagged TIP-1 protein, including the wild type (WT) or a mutant (MUT) with a dysfunctional PDZ domain (H90A) [16], were kindly provided by Dr. Paul A. Welling at University of Maryland (Baltimore, MD). A recombinant plasmid encoding FLAG-tagged LZAP [12] was a generous gift from Dr. Wendell G. Yarbrough at Vanderbilt University (Nashville, TN). QuikChangeTM Site- Directed Mutagenesis Kit (Stratagene, Santa Cruz, CA) was used to create a LZAP mutant at a putative C-terminal PDZ-binding motif (from -LMGTSL to-LMGASA, the mutations are under- lined) with primers 59-CGGAAGCTTGGTACCATGGAG-39 and 59-CCGTCTAGATCAGGCAGAGGCTCCCAT- CAGGTTCAC-39 (Sigma). All the constructs were confirmed with DNA sequencing. GST or GST-tagged TIP-1 proteins were expressed in E. coli and purified to homogeneity as described [25]. RNA interference and cell transfection Constructs expressing small hairpin RNA (shRNA) were purchased from Open Biosystems (Huntsville, AL). Two TIP-1 specific shRNAs (TIP-1 #1: 59-GGCTAACAGCTGATCCCAA- 39 and TIP-1 #2: 59-GCAAAGAGTTGAAATTCACAA-39) match with different region of human and mouse TIP-1 mRNA transcripts. A validated shRNA control with no obvious matched sequence in human and mouse genome was also used. Small Interfering RNA (siRNA) for the LZAP knockdown were designed according to the published data [13] and synthesized by Sigma. Three LZAP siRNAs used in this study include CS3-1: r(GCAGAUUGCCAAGUGCCAGC), CS3-2: r(GCAGGA- GAUUAUAGCUCUGUA), and CS3-3: r(GAGGCCUCCAC- GAAGAAUAUU). A control siRNA was purchased from Qiagen (Germantown, MD). Interfering RNA pool was used for silence p53 (siRNA-1: GGAGAAUAUUUCACCCUUC; siRNA-2: GCAGUCAGAUCCUAGCGUC; siRNA-3: GUGCAGCU- GUGGGUUGAUU; and siRNA-4: GAAAUUUGCGUGUG- GAGUA. Thermo Scientific Inc., Waltham, MA). RNAiMAX (Invitrogen) was used for the siRNA transfection by following the manufacturer’s instructions. Transfection of cells with the recom- binant plasmids and the establishment of stable clones were conducted with standard protocols [27]. The protein expression level was detected by western blot analysis of whole cell lysate with the specific antibodies. Clonogenic assay and detection of apoptosis Cells were disaggregated, counted with hemocytometer, plated on new plates with fresh medium and allowed to attach for around 5 hours. The cells were then irradiated at 0 (mock treatment), and variable doses of X-ray, respectively. After 10 to 14 days, colonies were counted after staining with 1% methylene blue. Cell survival fractions were calculated to determine dose modification factor (DMF) upon the dose ratio at the survival fraction of 0.1 [25]. To detect cell apoptosis, the cells exposed to the mock treatment or 10 Gy X-ray were collected at 24 or 48 hours post treatment. Cell apoptosis was detected by western blot analyses of the cleaved caspase-3 within cell lysates. The blots were scanned using a densitometer to quantify the proteins of interest. The semi- quantitative measurements were sequentially normalized to actin controls and those from the cells transfected with a respective vector control (counted as 1). Cells were also stained with Annexin PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45402