Expression of TIP-1 Confers Radioresistance of Malignant Glioma Cells

<div><h3>Background</h3><p>Malignant gliomas represent one group of tumors that poorly respond to ionizing radiation (IR) alone or combined with chemotherapeutic agents because of the intrinsic or acquired resistance. In this study, TIP-1 was identified as one novel protein that confers resistance of glioma cells to IR.</p> <h3>Methodology/Principal Findings</h3><p>Meta-analysis indicated that high TIP-1 expression levels correlate with the poor prognosis of human malignant gliomas after radiotherapy. Studies with established human glioma cell lines demonstrated that TIP-1 depletion with specific shRNAs sensitized the cells to IR, whereas an ectopic expression of TIP-1 protected the glioma cells from the IR-induced DNA damage and cell death. Biochemical studies indicated that TIP-1 protein promoted p53 ubiquitination and resulted in a reduced p53 protein level. Furthermore, p53 and its ubiquitination are required for the TIP-1 regulated cellular response to IR. A yeast two-hybrid screening identified that TIP-1, through its single PDZ domain, binds to the carboxyl terminus of LZAP that has been studied as one tumor suppressor functioning through ARF binding and p53 activation. It was revealed that the presence of TIP-1 enhances the protein association between LZAP and ARF and modulates the functionality of ARF/HDM2 toward multi-ubiquitination of p53, while depleting TIP-1 rescued p53 from polyubiquitination and degradation in the irradiated glioma cells. Studies with a mouse xenograft model indicated that depleting TIP-1 within D54 cells improved the tumor growth control with IR.</p> <h3>Conclusions/Significance</h3><p>This study provided the first evidence showing that TIP-1 modulates p53 protein stability and is involved in the radioresistance of malignant gliomas, suggesting that antagonizing TIP-1 might be one novel approach to sensitize malignant gliomas to radiotherapy.</p> </div

LZAP is required for the TIP-1-regulated IR-induced apoptosis and p53 polyubiquitination in D54 glioma cells.

<p><i>A)</i> Western blot analysis of LZAP knockdown in D54 glioma cells with siRNAs. Whole cell lysates were analyzed at 72 hours post the transfection. A validated control siRNA was included. <i>B)</i> Western blot analysis and quantification of caspase-3 cleavage with or without LZAP knockdown. Cells were transfected with LZAP-targeting siRNA or a control siRNA 72 hours prior to irradiation at 5 Gy. Whole cell lysates were analyzed at 24 hours post IR treatment. Significance was determined by comparing the denoted group to the respective vector control. *<i>p</i><0.05; ** <i>p</i>>0.05 with t-test. <i>C)</i> Western blot analysis of the p53 protein levels and ubiquitination with or without LZAP knockdown. The cells were irradiated at 5 Gy. MG132 was added at 8 hours post IR treatment and incubated for 4 more hours before the cell lysates were collected to analyze the p53 ubiquitination. The representative image shows the ubquitinated p53 ladder (pointed with arrows) as well as overall LZAP, p53, TIP-1 and actin in each cell lysate.</p

TIP-1 facilitates DNA damage repair after IR treatment.

<p><i>A</i>) γ-H2AX foci staining. D54 cells were mock treated or irradiated at 5 Gy, and γ-H2AX foci were detected by immunofluorescence staining at 0.5, 4 and 24 hours post the treatment. The representative images show positive cells (pointed with arrow heads) containing more than 5 foci. <i>B)</i> Percentage of γ-H2AX foci-positive cells was calculated upon at least 100 cells from each of the triplicate experiments. <i>C)</i> Representative images (×400) of IR-induced DNA damage visualized with neutral comet assay. <i>D)</i> Percentage of cells with comet tails (as one indicator of popularity of DNA strand breaks among cells). <i>E)</i> Statistics of tail moments (as one indicator of DNA damage severity in individual cells). At least 75 comet images (including more than 100 cells) were analyzed for each group. Shown are the mean +/− standard deviation of at least three independent experiments. Significance was determined by comparing the denoted group to the respective vector control with the same treatment, respectively. * <i>p</i><0.05, t-test.</p

TIP-1 interacts with LZAP and enhances the protein interaction between LZAP and ARF within the irradiated D54 glioma cells.

<p><i>A)</i> Putative TIP-1 interacting proteins identified through yeast two-hybrid screening of a human fetal brain cDNA library. <i>B)</i> 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 <i>(C)</i> with recombinant GST-fused TIP-1 (WT) protein or a mutant (MUT) with a dysfunctional PDZ domain, as well as co-immunoprecipitation assay <i>(D)</i> by using D54 cells that were co-transfected with recombinant plasmids as indicated. <i>E)</i> 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.</p

TIP-1 expression levels correlate with the survival probability of patients with gliobmatoma multiforme.

<p>Patients with elevated TIP-1 expression levels (log2 tumor/normal ratio >1.5) are defined as “TIP-1 high” group, those with the ratio less than 0.5 are defined as “TIP-1 low” group. Kaplan-Meier survival probability curves were developed with MedCalc. Statistical analysis was conducted with Log Rank Test (<i>p</i><0.05).</p

TIP-1 knockdown sensitizes glioma cells to ionizing radiation.

<p><i>A</i>) 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. <i>B)</i> 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.</p

TIP-1 suppressed p53 activation after irradiation.

<p>D54 cells with an ectopic expression <i>(A)</i> or shRNA depletion <i>(B)</i> were mock treated or irradiated at 5 Gy, whole cell lystates were collected at the indicated time points post the treatment for western blot analyses to profile pathway mediators that sense, transduce, or execute cellular response to DNA damage. <i>C)</i> Semi-quantification of p53 upon the band density on blots using a densitometer. Significance was determined by comparing the denoted group to the respective vector control with the same treatment, respectively. *<i>p</i><0.05, t-test. <i>D)</i> Analyses of p53 ubiquitination. Cells were mock treated or with IR (5 Gy). MG132 was added at 8 hours post the treatment and incubated for 4 more hours before the cell lysates were collected to analyze the p53 ubiquitination. The ubiquitinated p53 protein was immunoprecipitated with antibody against p53 and blotted with ubiquitin-antibody after the proteins were separated with SDS-PAGE. The representative image shows the ubquitinated p53 ladder (pointed with arrows), as well as overall p53, TIP-1 and actin in each cell lysate.</p

TIP-1 protein levels correlate to the radiation-induced apoptosis in D54 cells.

<p><i>A)</i> Caspase-3 cleavage within D54 cells were examined with western blot. Cells received mock (untreated) or 5 Gy IR treatment were analyzed 24 hours post the treatment. The blots were scanned using a densitometer to quantify the proteins of interest. Relative fold change of caspase-3 activation for each cell line was normalized to the respective vector control (counted as 1). <i>B)</i> Flow cytometric analyses of Annexin V/PI stained cells. Cells were analyzed at the indicated time points post the mock or 10 Gy IR treatment. Shown are mean +/− standard deviation of at least three independent experiments performed in triplicate. Significance was determined by comparing the denoted group to the respective vector control with the same treatment, respectively. * <i>p</i><0.05 with t-test.</p

Schemetic illustration of the putative roles of TIP-1 in the radioresistance of malignant gliomas.

<p>Schemetic illustration of the putative roles of TIP-1 in the radioresistance of malignant gliomas.</p

p53 mediates the TIP-1-regulated cellular response to IR of glioma cells.

<p><i>A)</i> 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. <i>B)</i> 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. * <i>p</i><0.05, ** <i>p</i>>0.05, all tests of significance were determined with a two-sided Student's <i>t</i> test. <i>C)</i> 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 µM 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.</p