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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.

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.