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Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma.Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity.Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity.Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

Supplementary Figures S1-S6; Supplementary Table S1.

DNA methylome analysis of glioblastoma (GBM) identified the HIV-1 Tat interactive protein 2 (HTATIP2) gene as aberrantly methylated and silenced. HTATIP2 is a negative regulator of importin β-mediated (KPNB1) cytoplasmic-nuclear translocation of proteins and its deregulation may alter the functionality of cancer relevant nuclear proteins. We propose N-Methylpurine-DNA Glycosylase (MPG), responsible for removing alkylated bases and initiating base excision repair (BER), as a potential GBM relevant candidate. Here we investigated the role of epigenetic silencing of HTATIP2 on the subcellular localization of MPG, and MPG-mediated DNA repair. Induction of HTATIP2 expression in GBM cells lead to a significant shift of predominantly nuclear to cytoplasmic MPG, while depletion of endogenous levels of HTATIP2 resulted in enhanced nuclear MPG localization. We observed exclusion of MPG from the area exhibiting co-localization of HTATIP2 and KPNB1 in proximity to the nuclear membrane, suggesting competition of HTATIP2 with MPG to bind to KPNB1. In accordance, pharmacologic inhibition of KPNB1 similarly induced cytoplasmic retention of MPG as HTATIP2 expression. Reduced nuclear MPG localization, induced by HTATIP2 expression or depletion of MPG, yielded less P-H2AX-positive cells upon treatment with an alkylating agent. This suggested reduced MPG-mediated formation of apurinic/apyrimidinic (AP) sites, leaving behind unrepaired DNA lesions, hence, reflecting a reduced capacity of BER in response to the alkylating agent. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and eventually contributing to treatment resistance.

In a genome-wide DNA methylation analysis of glioblastoma we identified aberrant methylation of the HIV-1 Tat interactive protein 2 (HTATIP2) gene promotor. This was strongly correlated with downregulation of HTATIP2 gene expression, suggesting a potential tumor suppressor function in glioblastoma. HTATIP2 has been shown to inhibit nuclear translocation through interaction with β-importins. We hypothesize that deregulation of HTATIP2 expression inhibits nuclear entry of cancer-relevant proteins, thereby disturbing their specific function in the nucleus. We identified N-Methylpurine-DNA Glycosylase (MPG) as a potential cancer relevant candidate with the observation of nuclear and/or cytoplasmic localization in glioblastoma. MPG is a DNA repair protein that recognizes DNA lesions (N7-meG, N3-meA) and initiates base excision repair. We have shown that nuclear MPG expression contributes to resistance of glioblastoma to treatment with the alkylating agent temozolomide. Here we investigated the effect of HTATIP2 on cellular localization of MPG using (i) an inducible system (TET-ON) to express HTATIP2 in non-expressing glioblastoma cells, and (ii) HTATIP2-targeting siRNAs to knock down HTATIP2 in cells with endogenous expression. Results from confocal microscopy or Imagestream analyses showed a significant cytoplasmic retention of MPG in presence of HTATIP2, while knock-down of HTATIP2 resulted in nuclear MPG localization. Cytoplasmic retention of MPG in the presence of HTATIP2 was associated with a significant increase in γ-H2Ax signal after treatment with the alkylating agent: methyl methanesulfonate (MMS), suggesting increase in DNA damage. Mechanistically, we found that HTATIP2 co-localizes with importin β1, and excludes MPG localization. Furthermore, HTATIP2 displayed a similar effect on cytoplasmic retention of MPG as pharmacologic inhibition of Importin β 1. Taken together, these results suggest that epigenetic silencing of HTATIP2 may increase nuclear localization of MPG, thereby increasing the capacity of the tumor cells to repair treatment related lesions and thereby contributing to treatment resistance.

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception ( Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255–66. ©2010 AACR.

N6-O′2-dibutyryl cAMP (dbcAMP), N6-monobutyryl cAMP (N6-mbcAMP), 8-Chloro cAMP (ClcAMP), and O′2-monobutyryl cAMP (O′2-mbcAMP) were used to study glial fibrillary acidic protein (GFAP) induction in rat C6 glioma. With the exception of O′2-mbcAMP, these cAMP analogs induced GFAP after stimulation of cells with a concentration of 0.5-1 mM. Only dbcAMP and N6-mbcAMP increased the intracellular concentration of cAMP. Protein kinase A (PKA) activation is often proposed to be involved in GFAP expression in astrocytes. Ion-exchange chromatography indicated that protein kinase activity is associated with PKA type II in C6. dbcAMP, N6-mbcAMP, and ClcAMP upregulated the amount of cAMP-binding proteins approximately twofold. RI was upregulated in the cytosol and particulate fraction, whereas RII was not affected after stimulation with dbcAMP. Concomitant, the PKA activity decreased approximately 60% and 40% in the cytosol and particulate fraction, respectively. CREB is constitutively expressed in C6 and is downregulated after stimulation with dbcAMP. The membrane-permeable PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) did not suppress the induction of GFAP-mRNA and its translation into GFAP. On the contrary, depending on the time difference between H89 and dbcAMP addition to C6, GFAP synthesis could even be potentiated more than twofold. Experiments in the presence of cycloheximide showed that protein synthesis is necessary for GFAP transcription. Although all components of the PKA signal transduction pathway are present in C6, GFAP synthesis is not dependent on PKA activation but required the synthesis of an unidentified factor. J. Neurosci. Res. 48:324–333, 1997. © 1997 Wiley-Liss, Inc.

Nucleoside diphosphate kinase (NDPK/nm23) ATP/ GDP phosphotransferase activity and serine autophosphoryla- tion is inhibited by M-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6- mbcAMP is a substrate of the enzyme. DbcAMP and O'2- mbcAMP, cAMP analogues with a modified 0'2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed. 2.3. Serine autophosphorylation of NDPK Autophosphorylation was performed in buffer B (25 mM HEPES (pH 7.3), 140 mM NaCl, 1 mM MgCl2, 0.8 mM CaCl2, 5 mM KC1, 5 mM EDTA) and in the presence of 20 nM fr 32P)ATP (4500 Ci/ mmol). Incubation was at 37°C for 30 min. The reaction was stopped by addition of SDS-PAGE sample buffer to a final concentration of 0.5% (w/v) SDS, 10% (v/v) glycerol, 0.5% (v/v) 2-mercaptoethanol, 6 mM Tris-HCl (pH 6.8). After boiling of the samples for 5 min, they were analyzed by SDS-PAGE on a 20% (w/v) polyacrylamide gel. The gel was Coomassie stained (30 min in 0.1% (w/v) CBB-R250, 7.5% (v/v) acetic acid, 40% (v/v) methanol) and destained for 16 h by successive washes in 10% (v/v) acetic acid, 40% (v/v) methanol. After dehydration in 50% ethanol the gel was dried and autoradiographed.