Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O 6 -methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O 6 -alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X L . We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r 2 ¼ 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O 6 -benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X L protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ. Keywords: BCL-X L , glioblastoma, O 6 -methylguanine DNA methyltransferase, p53, temozolomide. Temozolomide (TMZ) is a cytotoxic alkylating agent which has shown activity in recurrent anaplastic glioma and glioblastoma (Yung et al. 1999(Yung et al. , 2000. Moreover, the recent European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada trial on concomitant and adjuvant TMZ in addition to radiotherapy as the first-line treatment of glioblastoma produced an increase in median survival from 12.1 to 14.6 months and an increase in the 2-year survival rate from 10 to 26% compared with radiotherapy alone (Stupp et al. 2005).Temozolomide is rapidly and completely absorbed after oral administration and undergoes spontaneous hydrolysis at physiological pH to its active metabolite 5-(3-methyltriazeno)-imidazole-4-carboxamide. 5-(3-methyltriazeno)-imidazole-4-carboxamide causes DNA damage by methylation of the O 6 position of guanine. However, DNA adducts different from O 6 -methylguanine might also be involved in the cytotoxicity induced by methylating agents and poly (ADP-ribose) polymerase (PARP) inhibitors may also sensitize tumour cells to TMZ-induced cell death (Tentori e...

Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-X L conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosisprotein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.

Epidermal growth factor receptor (EGFR) signaling has become an important target for drug development because EGFR signaling enhances tumor cell proliferation, migration, and invasion and inhibits apoptosis. However, the results of clinical trials using EGFR inhibitors in patients with solid tumors have been disappointing. Here, we report a protective effect of the EGFR inhibitors AG1478 and PD153035 against cell death induced by acute hypoxia, which contrasts with their proapoptotic effects under normoxia. Under hypoxic conditions, both agents reduced glucose consumption, delayed ATP depletion, and preserved the mitochondrial membrane potential. Exposure either to hypoxia or the EGFR inhibitors under normoxic conditions resulted in the dephosphorylation of ribosomal protein S6, a player in the energy and nutrient-sensing pathway governed by mammalian target-of-rapamycin (mTOR). Combined inhibition of phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase-1/2 (ERK1/2) mimicked the protective effects of EGFR inhibition on hypoxia-induced cell death and protein S6 dephosphorylation. These results caution that therapies targeting EGFR signaling pathways can protect tumor cells from acute hypoxia.

APRIL (a proliferation-inducing ligand)

No abstract

Death ligands such as CD95 ligand (CD95L) have limited activity against glioma cells under normoxic conditions. Hypoxia is a critical aspect of the microenvironment of gliomas in vivo. We investigated the effect of co-exposure to acute hypoxia and CD95 ligand in three human malignant glioma cell lines with different susceptibility to CD95L under normoxic conditions. Hypoxia sensitized all three cell lines towards CD95L-induced cell death. Co-exposure resulted in apoptotic changes in the early phase, with gradual conversion to secondary necrosis with increasing length of hypoxia. The mitochondrial injury induced by hypoxia was enhanced by co-treatment, and caspase cleavage became prominent. Inhibition of the epidermal growth factor receptor (EGFR), although sensitizing glioma cells to CD95L under normoxia, protects glioma cells from hypoxia by reducing energy consumption. However, the opposing effects of EGFR signalling on death induced by CD95L or hypoxia were neutralized by co-exposure to hypoxia and CD95L. Furthermore, inhibition of protein synthesis by cycloheximide also reduced glucose consumption and conferred protection from hypoxia, but did not modulate CD95L-induced cell death under hypoxic conditions. These results suggest that death ligands may be useful to target hypoxic tumour cells resistant to conventional therapies or to complement strategies aiming at the induction of tumour hypoxia.