Radiosensitivity Of Human Glioma Cells Research Articles

Regulation of a novel circATP8B4/miR-31-5p/nestin ceRNA crosstalk in proliferation, motility, invasion and radiosensitivity of human glioma cells.

Deregulation of circular RNAs (circRNAs) is frequent in human glioma. Although circRNA ATPase phospholipid transporting 8B4 (circATP8B4) is highly expressed in glioma, its precise action in glioma development is still not fully understood. The relationship of microRNA (miR)-31-5p and circATP8B4 or nestin (NES) was predicted by bioinformatic analysis and confirmed by RNA pull-down and Dual-luciferase reporter assays. CircATP8B4, miR-31-5p and NES were quantified by qRT-PCR or western blot. Cell functional behaviors were assessed by EdU, wound-healing and transwell invasion assays. Xenograft model experiments were performed to define circATP8B4's activity in vivo. CircATP8B4, a true circular transcript, was upregulated in human glioma. CircATP8B4 downregulation weakened glioma cell growth, motility, and invasion and facilitated radiosensitivity. Mechanistically, circATP8B4 and NES 3'UTR harbored a shared miR-31-5p pairing site, and circATP8B4 involved the post-transcriptional NES regulation by functioning as a competing endogenous RNA (ceRNA). Furthermore, the miR-31-5p/NES axis participated in circATP8B4's activity in glioma cell proliferation, motility, invasion and radiosensitivity. Additionally, circATP8B4 loss diminished tumor growth and enhanced the anticancer effect of radiotherapy in vivo. We have uncovered an uncharacterized ceRNA cascade, circATP8B4/miR-31-5p/NES axis, underlying glioma development and radiosensitivity. Targeting the ceRNA crosstalk may have potential to improve the outcome of glioma patients.

Using Hyperbaric Oxygen to Improve the Radiosensitivity of Human U251 Glioma Cells.

The aim of this study was to explore the use of hyperbaric oxygen to enhance the radiosensitivity of human glioma cells. Sub-cultured U251 human glioma cells were randomly divided into four groups: an untreated control group, cells treated with hyperbaric oxygen (HBO) only, cells treated with X-ray irradiation (X-ray) only, and cells treated with both HBO and X-ray. Cell morphology, cell proliferation activity, cell cycle distribution, and apoptosis were observed in these groups to evaluate the role of HBO in improving the radiosensitivity of glioma cells. With the increase in X-ray doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy), the survival fraction (SF) of glioma cells gradually decreased. Significantly lower SF was observed for the cells treated with the HBO and X-ray together than in the X-ray group for each dose (all P < 0.05). The proliferation inhibition was significantly higher in the HBO combined with X-ray group than in the X-ray group for each dose (all P < 0.05) for the U251 cell line. The percentage of G2/M phase cells was significantly higher in the HBO combined with X-ray (2 Gy) group (26.70% ± 2.46%) and the HBO group (22.36% ± 0.91%) than in the control group (11.56% ± 2.01%) and X-ray (2 Gy) group (10.35% ± 2.69%) (all P < 0.05). U251 cell apoptosis was significantly higher in the HBO combined with X-ray (2 Gy) group than in the HBO group, the X-ray (2 Gy) group, and the control group (all P < 0.05). We conclude that HBO can enhance the proliferation inhibition and apoptosis of glioma U251 cells by blocking glioma cells in the G2/M phase and improve the radiosensitivity of U251 glioma cells.

Enhancement of radiotherapy efficacy by silver nanoparticles in hypoxic glioma cells

Radiotherapy is one of the most widely used treatments for therapy of malignant tumors, but resistance to radiation of hypoxic cells in tumor tissues is still a serious concern. Previous studies have demonstrated that silver nanoparticles (AgNPs) enhance the radiosensitivity of human glioma cells in vitro, but the effect of AgNPs on hypoxic glioma cells has not been investigated in detail. The main purpose of this study is to evaluate the radiosensitizing efficacy of AgNPs on hypoxic glioma cells. The half maximal inhibitory concentration (IC50) values of AgNPs for the hypoxic U251 cells and C6 cells were 30.32 μg/mL and 27.53 μg/mL, respectively. The sensitization enhancement ratio (SER) demonstrated that AgNPs exhibit higher capacity in radiosensitization in hypoxic cells (U251: 1.78; C6: 1.84) than that in normoxic cells (U251: 1.34; C6: 1.45). The underlying mechanism of AgNPs’ radiosensitization in hypoxic cells is through the promotion of apoptosis and enhanced destructive autophagy. There is evidence of crosstalk between apoptosis and autophagy in AgNPs-radiosensitized hypoxic cells where inhibition of autophagy results in decreased apoptosis. These findings suggest that AgNPs can be used as a highly effective nano-radiosensitizer for the treatment of hypoxic glioma.

Role of H-Ferritin in Radiosensitivity of Human Glioma Cells

Role of H-Ferritin in Radiosensitivity of Human Glioma Cells

Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage.

Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro. Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy. Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2. Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.

Radiosensitization of human glioma cells by tamoxifen is associated with the inhibition of PKC-ι activity in vitro.

The present study aimed to investigate the radiosensitizing effects of tamoxifen (TAM), a non-steroidal anti-estrogen drug, in human glioma A172 and U251 cells in vitro. A colony-forming assay revealed that TAM enhances radiosensitivity in A172 and U251 cells. Treatment with TAM also increased the percentage of apoptotic cells subsequent to ionizing radiation, and increased the expression of apoptotic markers, including cleaved caspase-3 and poly(ADP-ribose) polymerase. Ionizing radiation induced G2/M phase arrest, which was alleviated within 24 h when the radiation-induced DNA damage was repaired. However, flow cytometry analysis revealed that TAM treatment delayed the recovery of cell cycle progression. Additional examination demonstrated that TAM-mediated protein kinase C-ι (PKC-ι) inhibition may lead to the activation of pro-apoptotic B-cell lymphoma 2-associated death promoter, and the dephosphorylation of cyclin-dependent kinase 7, resulting in increased cell apoptosis and sustained G2/M phase arrest following exposure to radiation. The present data indicate that the radiosensitizing effects of TAM on glioma cells are partly due to the inhibition of PKC-ι activity in vitro.

CpG ODN107 potentiates radiosensitivity of human glioma cells via TLR9-mediated NF-κB activation and NO production

Radiotherapy is a standard treatment for glioma patient with or without surgery; radiosensitizer can increase tumor sensitivity for radiotherapy. Herein, a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN107) as a radiosensitizer was investigated in vitro and in vivo, and the possible mechanisms were studied in vitro. In the present experiments, the human glioma U87 cell line used herein was resistant to 5 Gy of β-ray irradiation. The results showed that 10 μg/ml of CpG ODN107 in combination with irradiation significantly inhibited cell proliferation both in MTT assay and colony formation experiments. Tumor growth was inhibited by CpG ODN107 in combination with local irradiation but not by local irradiation or CpG ODN107 alone in human glioma xenograft model in nude mice. The inhibition ratio of tumor growth produced by CpG ODN107 (1.7, 5, and 15 mg/kg) in combination with irradiation was 27.3, 67.0, and 65.5 %, respectively. Further molecular mechanisms were studied in vitro. The results showed that the expressions of iNOS, NO, TLR9 mRNA, and NF-κB p50/p65 increased in the cells treated with CpG ODN107 in combination with irradiation. CpG ODN107 in combination with irradiation did not induce apoptosis but induced cell cycle arrest at G(1) phase. The said results demonstrated that CpG ODN107 possessed a radiosensitizing effect via TLR9-mediated NF-κB activation and NO production in the tumor cells, leading to cell cycle arrest. Therefore, CpG ODN107 is a potential candidate as radiosensitizer for human glioma.

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.

Effects of LRIG1 expression on radiosensitivity of human glioma cells and the possible mechanism

Objective To investigate the effect of the expression of leucine-rich repeats and immunoglobulin-like domains 1 （LRIG1） on the radiosensitivity of human glioma cell line U251 cells and the possible mechanism. Methods The PEGFP-N1, PEGFP-LRIG1 plasmids were transfected into U251 cells with application of liposome-mediated gene transfer technology. And the cells stably transfected were selected by G418. The radiosensitivity of N1-U251 and LRIG1-U251 cells was analyzed by clonogenic assay. Western blotting demonstrated the expression of LRIG1 and RAD51. Repairment of double-strand （ds） DNA breaks was measured by comet assay. Results The LRIG1 protein level in LRIG1-U251 group （0. 352±0. 011 ） was significantly up-regulated compared to NI-U251 group （0. 071±0. 003 ） （ P 〈 0. 01 ）. Overexpression of LRIG1 up-regulated the radiosensitivity of U251 cells with the sensitive enhancement ratio （SER） being 1. 448. The RAD51 protein levels were significantly suppressed before （P 〈0.01 ） and after irradiation （P 〈 0.01） by up-regulating the LRIG1 expression. Comet assay demonstrated the overexpression of LRIG1 increased the Olive Tail Moment of U251 cells from 21.14±3.42 to 32. 81±5.61 after 8-h radiation （P 〈 0.05）. Conclusion Up-regulation of the LRIG1 expression can down-regulate the RAD51 expression, and then inhibit the repairment of dsDNA breaks, increase the radiosensitivity of U251 cells. Key words: Glioma; Leucine-rich repeats and immunoglobulin-like domains 1; RAD51; Radiosensitivity

Enhanced radiosensitization of human glioma cells by combining inhibition of poly(ADP-ribose) polymerase with inhibition of heat shock protein 90

Glioblastoma multiforme (GBM) are the most common primary brain tumor and are resistant to standard therapies. The nondividing nature of normal brain provides an opportunity to enhance the therapeutic ratio by combining radiation with inhibitors of replication-specific DNA repair pathways. Based on our previous findings that inhibition of poly(ADP-ribose) polymerase (PARP) increases radiosensitivity of human glioma cells in a replication-dependent manner and generates excess DNA breaks that are repaired by homologous recombination (HR), we hypothesized that inhibition of HR would amplify the replication-specific radiosensitizing effects of PARP inhibition. Specific inhibitors of HR are not available, but the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been reported to inhibit HR function. The radiosensitizing effects of 17-AAG and the PARP inhibitor olaparib were assessed, and the underlying mechanisms explored. 17-AAG down-regulated Rad51 and BRCA2 protein levels, abrogated induction of Rad51 foci by radiation, and inhibited HR measured by the I-Sce1 assay. Individually, 17-AAG and olaparib had modest, replication-dependent radiosensitizing effects on T98G glioma cells. Additive radiosensitization was observed with combination treatment, mirrored by increases in gammaH2AX foci in G(2)-phase cells. Unlike olaparib, 17-AAG did not increase radiation sensitivity of Chinese hamster ovary cells, indicating tumor specificity. However, 17-AAG also enhanced radiosensitivity in HR-deficient cells, indicating that its effects were only partially mediated by HR inhibition. Additional mechanisms are likely to include destabilization of oncoproteins that are up-regulated in GBM. 17-AAG is therefore a tumor-specific, replication-dependent radiosensitizer that enhances the effects of PARP inhibition. This combination has therapeutic potential in the management of GBM.

Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence of this effect and its therapeutic potential. KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in gammaH2AX and Rad51 foci. The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.

Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells

This study investigates the hypothesis that CuZn superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and the G 2/M-checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0–10 Gy) and assayed for cell survival, cellular ROS levels, cell-cycle-phase distributions, and cyclin B1 expression. SOD1-overexpressing cells were radioresistant compared to wild-type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (approximately twofold) in DHE fluorescence beginning at 2 days postirradiation, which remained elevated at 8 days postirradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability and an increase in the percentage of cells with sub-G 1 DNA content. SOD1 overexpression enhanced radiation-induced G 2 accumulation within 24 h postirradiation, which was accompanied by a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after radiation exposure a “metabolic redox response” regulates radiosensitivity of human glioma cells.

Temozolomide-Mediated Radiosensitization of Human Glioma Cells in a Zebrafish Embryonic System

The zebrafish (Danio rerio) is a popular vertebrate model for biomedical research. The rapid development, transparency, and experimental accessibility of the embryo offer opportunities for assessing the developmental effects of anticancer treatment strategies. We therefore systematically investigated parameters for growing U251 human glioma cells expressing red fluorescent protein (U251-RFP) in zebrafish embryos. Factors optimized include injection volume, number of cells injected, anatomic site of injection, age of the embryo at the time of injection, and postinjection incubation temperature. After injection into the embryos, the U251-RFP cells proliferated and the resultant tumors, and even individual cells, could be visualized in real-time via fluorescence microscopy without the need for sacrifice. These tumors recruited host zebrafish vasculature, suggesting cancer cell-host tissue interactions. Having optimized parameters for introducing and growing these human cells in the zebrafish embryos, we exposed both embryos and transplanted cancer cells to ionizing radiation and temozolomide, either alone or in combination. The human tumors in each embryo were substantially diminished following exposure to ionizing radiation and the decrease was further enhanced by pretreatment with temozolomide. In contrast, temozolomide had no discernible effects on embryonic development. These results together support the relative safety of temozolomide during embryonic development, as well as its anticancer efficacy when combined with radiation. These results suggest the value of the zebrafish model for in vivo testing of the efficacy and safety of anticancer strategies, especially on the very young.

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0-6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24-72 h exposure to 250-750 microM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 microM meloxicam for 24 h increased the fraction of cells in the radiosensitive G(2)/M cell cycle phase in D384 (18-27%) and U251 (17-41%) cells. 750 microM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: Independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration

Purpose: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines.Materials and methods: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 – 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis.Results: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 μM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 μM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 μM) or Celecoxib (50 μM) and U87 with NS-398 (200 μM) after radiation resulted even in radioprotection.Conclusions: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

Inhibition of Akt by the alkylphospholipid perifosine does not enhance the radiosensitivity of human glioma cells

Akt has been implicated as a molecular determinant of cellular radiosensitivity. Because it is often constitutively activated or overexpressed in malignant gliomas, it has been suggested as a target for brain tumor radiosensitization. To evaluate the role of Akt in glioma radioresponse, we have determined the effects of perifosine, a clinically relevant alkylphospholipid that inhibits Akt activation, on the radiosensitivity of three human glioma cell lines (U87, U251, and LN229). Each of the glioma cell lines expressed clearly detectable levels of phosphorylated Akt indicative of constitutive Akt activity. Exposure to a perifosine concentration that reduced survival by approximately 50% significantly reduced the level of phosphorylated Akt as well as Akt activity. Cell survival analysis using a clonogenic assay, however, revealed that this Akt-inhibiting perifosine treatment did not enhance the radiosensitivity of the glioma cell lines. This evaluation was then extended to an in vivo model using U251 xenografts. Perifosine delivered to mice bearing U251 xenografts substantially reduced tumor phosphorylated Akt levels and inhibited tumor growth rate. However, the combination of perifosine and radiation resulted in a less than additive increase in tumor growth delay. Thus, in vitro and in vivo data indicate that the perifosine-mediated decrease in Akt activity does not enhance the radiosensitivity of three genetically disparate glioma cell lines. These results suggest that, although Akt may influence the radiosensitivity of other tumor types, it does not seem to be a target for glioma cell radiosensitization.

Dihydroartemisinin enhances radiosensitivity of human glioma cells in vitro

The antimalarial agent, artemisinin, also confers cancer-specific cytotoxic effects by reacting with ferrous iron atoms to form free radicals. Here, we investigated the radiosensitizing effects of dihydroartemisinin on glioma cells and assessed some possible mechanisms for these effects. U373MG glioma cells treated with various concentrations of dihydroartemisinin plus radiation, and efficiency of radiosensitization was assessed by clonogenic survival assay. Expression and activity of antioxidant enzymes, glutathione-S-transferase (GST) were quantified by western blot and enzymatic activity analyses, respectively. Dihydroartemisinin showed higher cytotoxicity in the glioma cell lines than in the liver, breast or cervical cancer cell lines. In clonogenic survival assays, treatment with dihydroartemisinin alone dose-dependently reduced the number of U373MG colonies, while treatment with dihydroartemisinin plus gamma-irradiation showed far lower clonal survival than cultures treated with radiation or dihydroartemisinin alone. The radiosensitizing effect of dihydroartemisinin was blocked significantly by the free radical scavengers, NAC and TIRON, indicating association with dihydroartemisinin-induced ROS generation. In addition, the radiation-induced expression of endogenous GST was suppressed by treatment with dihydroartemisinin. The radiosensitizing effect of dihydroartemisinin was also markedly enhanced by the addition of holotransferrin Taken together, our results strongly suggest that dihydroartemisinin triggers production of ROS and inhibits GST activity, leading to effective and therapeutically relevant radiosensitization of human glioma cells.

Inhibition of Rho pathways induces radiosensitization and oxygenation in human glioblastoma xenografts

We previously demonstrated in vitro that inhibiting the biological pathways of the small GTPase Rho radiosensitizes the human glioma U87 cell line. The aim of this study was to determine if Rho might be involved in the control of in vivo radiosensitivity altogether by controlling cellular radioresistance and by modifying tumor microenvironment. We demonstrate here that the in vivo induction of the dominant negative of Rho, RhoBN19, in U87 xenografts induces a significant decrease of tumor cell survival after irradiation more important than the one we previously observed in vitro. This in vivo increased effect of RhoBN19 expression is due to the improvement of the tumor oxygenation associated with a significant decrease of the vessel density and of the metalloproteinase 2 (MMP2) expression. Moreover, in vitro RhoBN19 expression in U87 cells leads to the inhibition of MMP2 activity. Our results demonstrate for the first time that inhibiting Rho pathways modifies the in vivo radiosensitivity of human glioma cells by controlling intrinsic radioresistance, hypoxia and angiogenesis. These data strongly suggest that Rho should be a major determinant of cellular resistance to ionizing radiation.

1021 Poster Radiosensitivity of human glioma cells and relationship with glioma specific genetic alterations

1021 Poster Radiosensitivity of human glioma cells and relationship with glioma specific genetic alterations

Radiosensitization of human glioma cells in vitro and in vivo with acyclovir and mutant HSV-TK75 expressed from adenovirus

Purpose : We recently reported that an adenovirus-expressing mutant HSV-TK75 (AdCMV-TK75) radiosensitized rat syngeneic gliomas in combination with low concentrations of acyclovir (ACV) much more effectively than a virus expressing wild-type herpes simplex virus thymidine kinase (HSV-TK). In this report we have examined whether similar radiosensitizing effects are also seen with human glioma cells in vitro and in vivo. Methods and Materials : Human U87 MG glioma cells were transduced with AdCMV-TK75 and exposed to ACV followed by single-dose irradiation and colony-forming survival assays. Similarly, U87 MG xenografts were infused with AdCMV-TK75 or Adβgal control virus, followed by ACV administration and fractionated irradiation. Therapeutic efficacy was monitored by tumor growth. Results : U87 MG cells transduced with AdCMV-TK75 were significantly more sensitive to ACV (3 μM) than cells transduced with either wild-type HSV-TK or control virus. To determine whether human cells also demonstrate improved radiosensitization similar to that seen with rat glioma cells and tumors, we transduced U87 MG cells with either AdCMV-TK75, AdCMV-TK, expressing wild-type HSV-TK, or Adβgal and then treated the cells with 3 μM of ACV. Cells transduced with AdCMV-TK75 were significantly more radiosensitive (dose enhancement ratio [D 37]: 2.6) by colony-forming survival assay than cells transduced with either AdCMV-TK or Adβgal. Furthermore, we found that U87 MG xenografts infused with AdCMV-TK75 by slow positive pressure infusion were more radiosensitive after administration of ACV than tumors infused with Adβgal. A more dramatic result was achieved when fractionated irradiation was carried out concurrently with ACV administration, in which case AdCMV-TK75-treated tumors did not grow at all. Conclusions : These results demonstrate that transduction of human glioma cells in vitro and infusion of xenografts in vivo with AdCMV-TK75 and treatment with concentrations of ACV that can be achieved in vivo produce similar radiosensitization, as previously reported with rat glioma cells and intracerebral syngeneic tumors. In addition, concurrent treatment with ACV and radiation therapy is more efficient than when ACV is administered before radiation therapy.