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A critical challenge in cancer research is to identify genetic lesions that sensitize patients to chemotherapy. p53, which is mutated in nearly one-third to half of glioblastomas, may be such a lesion. In this paper, we demonstrate that p53 disruption dramatically sensitizes glioblastoma cells to DNA topoisomerase I inhibitor-mediated apoptosis. Using 19 glioblastoma cell lines, including 15 low-passage ex vivo cell lines derived from patients, as well as isogenic glioblastoma cells varying in p53 status, we show that clinically relevant levels of SN-38 potently induce cell cycle arrest and temporary senescence in glioblastoma cells with wild-type p53 while causing massive apoptosis in p53-deficient cells (P<0.0002). We demonstrate that glioblastoma cells with wild-type p53 proliferate when recultured in drug-free medium, whereas p53-deficient cells do not. We also show that p16 protein expression is neither necessary nor sufficient for initiation and/or maintenance of SN-38-induced arrest/senescence. These results indicate that p53 disruption has a dramatic effect on how glioblastoma cells process topoisomerase I inhibitor-mediated DNA damage.Oncogene (2004) 23, 1283-1290. doi:10.1038/sj.onc.1207244 [ABSTRACT FROM AUTHOR]

In this paper, we report that the chicken Epidermal Growth Factor Receptor (EGF-R), encoded by the proto-oncogene c-erbB, is comprised of 28 exons and spans over 75 Kb. The four previously identified domains which make up the extracellular ligand binding region of the receptor are coded for by two copies of a 300 amino acid repeat. We have demonstrated that the 3' end of each repeat coincides with the 3' end of the last exon making up the repeat. This alignment suggests that an exon-duplication may have occurred in the ligand-binding region of the gene. The transmembrane domain is encoded within a single exon and the exon boundaries of the catalytic domain closely match those defined by structural homology with other kinases. Along with the 54 chicken splice sites, the region 5' to the first exon was also sequenced. The proposed promoter region is greater than 70% GC, contains five repeats of the consensus Sp1 binding sequence and does not have a CCATT or TATA box. In addition to the presence of these characteristic housekeeping features, expression analysis confirms the promoter activity of this region and four sets of TCC repeats similar to those found in the human EGF-R promoter have been identified. To our knowledge this represents the first complete description of the exon-intron structure of the EGF-receptor family. The elucidation of the EGF-R exon structure provides insight into the domain evolution of the receptor-kinases and the mechanisms for the oncogenic conversion of EGF-R in erythroleukemias and glioblastomas.

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O6-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O6-benzylguanine showed that, in gliomas, the apoptotic signal originates from O6-methylguanine (O6MeG) and that repair of O6MeG by MGMT prevents apoptosis. We further demonstrate that O6MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O6MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O6MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by γH2AX formation. Glioma cells mutated in DNA-PKcs, which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.Oncogene (2007) 26, 186–197. doi:10.1038/sj.onc.1209785; published online 3 July 2006 [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme is an aggressive form of brain cancer that responds poorly to chemotherapy and is generally incurable. The basis for the poor response of this cancer to chemotherapy is not well understood. The atypical protein kinases C (PKCι and PKCζ) have previously been implicated in leukaemia cell chemoresistance. To assess the role of atypical PKC in glioblastoma cell chemoresistance, RNA interference was used to deplete human glioblastoma cells of PKCι. Transfection of cells with either of two different RNA duplexes specific for PKCι caused a partial sensitisation to cell death induced by the chemotherapy agent cisplatin. To screen for possible mechanisms for PKCι-mediated chemoresistance, microarray analysis of gene expression was performed on RNA from glioblastoma cells that were either untreated or depleted of PKCι. This identified sets of genes that were regulated either positively or negatively by PKCι. Within the set of genes that were negatively regulated by PKCι, the function of the gene coding for GMFβ, an enhancer of p38 mitogen-activated protein kinase (MAP kinase) signaling, was investigated further, as the p38 MAP kinase pathway has been previously identified as a key mediator of cisplatin cytotoxicity. The expression of both GMFβ mRNA and protein increased upon PKCι depletion, and this was accompanied by an increase in cisplatin-activated p38 MAP kinase signaling. Transient overexpression of GMFβ increased cisplatin-activated p38 MAP kinase signaling and also sensitised cells to cisplatin cytotoxicity. The increase in cisplatin cytotoxicity seen with PKCι depletion was blocked by the p38 MAP kinase inhibitor SKF86002. These data show that PKCι can confer partial resistance to cisplatin in glioblastoma cells by suppressing GMFβ-mediated enhancement of p38 MAP kinase signaling.Oncogene (2006) 25, 2909–2919. doi:10.1038/sj.onc.1209312; published online 12 December 2005 [ABSTRACT FROM AUTHOR]

We previously demonstrated that protein kinase C-?(PKC-?) mediates a phorbol 12-myristate-13-acetate (PMA)-induced proliferative response in human glioblastoma (GBM) cells. In this report, we show that PMA-stimulated activation of PKC-?in U-251 GBM cells resulted in activation of both Akt and the mammalian target of rapamycin (mTOR) signaling pathways and an increase in cell proliferation. Expression of a kinase dead PKC-?(PKC-?KR) construct reduced the basal and PMA-evoked proliferation of PKC-?-expressing U-251 GBM cells, as well as abrogated the PMA-induced activation of Akt, mTOR, and the mTOR targets 4E-BP1 and STAT-3. Treatment of cells with the PI-3 kinase inhibitor LY294002 (10?µM) or the mTOR inhibitor rapamycin (10?nM) also reduced PMA-induced proliferation and cell-cycle progression. Expression of a constitutively active PKC-?(PKC-??NPS) construct in a GBM cell line with no endogenous PKC-?(U-1242) also provided evidence that PKC-?targets the Akt and mTOR signaling pathways. Moreover, activation of 4E-BP1 and STAT-3 in both PMA-treated U-251 and PKC-??NPS-expressing U-1242 GBM cells was inhibited by rapamycin. However, activation of Akt, but not mTOR was inhibited by the PI-3 kinase inhibitor LY294002. This study identifies Akt and mTOR as downstream targets of PKC-?that are involved in GBM cell proliferation.Oncogene (2004) 23, 9062-9069. doi:10.1038/sj.onc.1208093 Published online 18 October 2004 [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) amplification and type III mutation (EGFRvIII), associated with constitutive tyrosine kinase activation and high malignancy, are commonly observed in glioblastoma tumors. The association of EGFR and EGFRvIII with caveolins was investigated in human glioblastoma cell lines, U87MG and U87MG-EGFRvIII. Caveolin-1 expression, determined by RT-PCR, real-time quantitative PCR and Western blot, was upregulated in glioblastoma cell lines (two-fold) and tumors (20-300-fold) compared to primary human astrocytes and nonmalignant brain tissue, respectively. U87MG-EGFRvIII expressed higher levels of caveolin-1 than U87MG. In contrast, the expression of caveolin-2 and -3 were downregulated in glioblastoma cells compared to astrocytes. A colocalization of EGFR, but not of EGFRvIII, with lipid rafts and caveolin-1 was observed by immunocytochemistry. Association of EGFR and EGFRvIII with caveolae, assessed in vitro by binding to caveolin scaffolding domain peptides and in vivo by immunocolocalization studies in cells and caveolae-enriched cellular fraction, was phosphorylation-dependent: ligand-induced phosphorylation of EGFR resulted in dissociation of EGFR from caveolae. In contrast, inhibition of the EGFRvIII constitutive tyrosine phosphorylation by AG1478 increased association of EGFRvIII with caveolin-1. AG1478 also increased caveolin-1 expression and reduced glioblastoma cell growth in a semi-solid agar. The evidence suggests that the phosphorylation-regulated sequestration of EGFR in caveolae may be involved in arresting constitutive or ligand-induced signaling through EGFR responsible for glial cell transformation.Oncogene (2004) 23, 6967-6979. doi:10.1038/sj.onc.1207911 Published online 26 July 2004 [ABSTRACT FROM AUTHOR]