Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors to chemo-and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to cradiation, has the capacity to repopulate the tumors and contribute to their malignant progression. c-radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by c-radiation in CD1331 GSCs. Irradiation of CD1331 cells induced autophagy within 24-48 hr and slightly decreased the viability of the cells. c-radiation induced a larger degree of autophagy in the CD1331 cells as compared with CD1332 cells and the CD1331 cells expressed higher levels of the autophagy-related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD1331 cells to c-radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD1331 GSCs to c-radiation. ' UICCKey words: autophagy; glioma stem cells; c-radiation; ATG5; ATG12 Glioblastomas (GBMs), the most frequent and aggressive primary brain tumors, are characterized by increased proliferation, resistance to chemotherapy and radiotherapy and invasion into the surrounding normal brain tissue. 1,2 Current treatments include surgery, radiation therapy and chemotherapy. 3,4 Unfortunately, the prognosis of patients with GBMs remains extremely poor and has not changed significantly during the past several years. 5,6 Therefore, novel therapeutic approaches are needed to improve the poor prognosis of these patients.Recently, a small subpopulation of CD1331 cancer stem cells has been identified in specimens of GBM. 7,8 These glioma stem cells (GSCs) express additional stem cell markers, exhibit selfrenewal and differentiation to glial and neuronal lineages, and can initiate xenograft tumors. 9,10 Cancer stem cells in various tumors, including the GSCs, have been implicated in the enhanced radioresistance and in the repopulation of tumors following these treatments. 10,11 Thus, delineating the molecular mechanisms underlying the increased resistance of these cells to anticancer therapies is of utmost importance.Autophagy is a cellular pathway involved in protein and organelle degradation. 12,13 This process is regulated by a series of autophagy-related genes (ATGs) and a number of signaling molecules such as mTOR, AKT, and class I and class III phosphatidylinositol 3-kinase. 14,15 Autophagy is frequently activated in tumor cells following anticancer therapies such as chemotherapeutic drugs 16,17 or g-irradiation 1...

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

The mechanism underlying the important role of protein kinase C␦ (PKC␦) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKC␦ in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKC␦ since the phosphorylation of Erk1/2 was inhibited by a PKC␦-KD mutant and PKC␦ small interfering RNA. We recently reported that phosphorylation of PKC␦ on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKC␦ tyrosine mutants, we found that the phosphorylation of PKC␦ on these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKC␦ was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKC␦. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKC␦ and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKC␦ on tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis. PKC␦2 is a novel PKC isoform that plays a major role in apoptosis in a cell-and stimulus-specific manner (1). PKC␦ has been reported to affect both the extrinsic and intrinsic apoptotic pathways and to mediate the apoptotic effect of various stimuli such as etoposide (2, 3), oxidative stress (4), ceramide (5), cisplatin (6), and phorbol 12-myristate 13-acetate (7). Conversely, it has been recently recognized that PKC␦ can act as an anti-apoptotic kinase in some cellular systems including Sindbis virus-infected (8) and TRAIL-treated glioma cells (9), nitric oxide-induced macrophage cell death (10), and cells expressing activated p21 RAS (11). Important factors that regulate the apoptotic function of PKC␦ are phosphorylation on distinct tyrosine residues and its subcellular localization (1). Tyrosine phosphorylation of PKC␦ is now recognized as a critical determinant in the activation, cleavage, localization and substrate affinity of this isoform (12-16). In addition to the tyrosine phosphorylation of PKC␦ by phorbol 12-myristate 13-acetate and various growth factors (12,17,18), PKC␦ undergoes phosphorylation in response to many apoptotic stimuli including etoposide (2), TRAIL (9), oxidative stress (4, 19), ␥-radiation (20), and cisplatin (13). PKC␦ has been shown to activate multiple signaling pathways that are associated with cel...

Many anticancer drugs target the genomic DNA of cancer cells by generating DNA damage and inducing apoptosis. DNA repair protects cells against DNA damage-induced apoptosis. Although the mechanisms of DNA repair and apoptosis have been extensively studied, the mechanism by which DNA repair prevents DNA damage-induced apoptosis is not fully understood. We studied the role of the antiapoptotic Bcl-x(L) protein in nucleotide excision repair (NER)-facilitated cell protection against cisplatin-induced apoptosis. Using both normal human fibroblasts (NF) and NER-defective xeroderma pigmentosum group A (XPA) and group G (XPG) fibroblasts, we demonstrated that a functional NER is required for cisplatin-induced transcription of the bcl-x(l) gene. The results obtained from our Western blots revealed that the cisplatin treatment led to an increase in the level of Bcl-x(L) protein in NF cells, but a decrease in the level of Bcl-x(L) protein in both XPA and XPG cells. The results of our immunofluorescence staining indicated that a functional NER pathway was required for cisplatin-induced translocation of NF-kappaB p65 from cytoplasm into nucleus, indicative of NF-kappaB activation. Given the important function of NF-kappaB in regulating transcription of the bcl-x(l) gene and the Bcl-x(L) protein in preventing apoptosis, these results suggest that NER may protect cells against cisplatin-induced apoptosis by activating NF-kappaB, which further induces transcription of the bcl-x(l) gene, resulting in an accumulation of Bcl-x(L) protein and activation of the cell survival pathway that leads to increased cell survival under cisplatin treatment.