RESUMO
BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.
Assuntos
Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Gradação de Tumores , RNA Interferente Pequeno/genéticaRESUMO
ProP, an osmoprotectant symporter from the major facilitator superfamily was expressed, purified and reconstituted into proteoliposomes that are amenable to structural characterization using infrared spectroscopy. Infrared spectra recorded in both (1)H(2)O and (2)H(2)O buffers reveal amide I band shapes that are characteristic of a predominantly alpha-helical protein, and that are similar to those recorded from the well-characterized homolog, lactose permease (LacY). Curve-fit analysis shows that ProP and LacY both exhibit a high alpha-helical content. Both proteins undergo extensive peptide hydrogen-deuterium exchange after exposure to (2)H(2)O, but are surprisingly thermally stable with denaturation temperatures greater than 60 degrees C. 25-30% of the peptide hydrogens in both ProP and LacY are resistant to exchange after 72 h in (2)H(2)O at 4 degrees C. Surprisingly, these exchange resistant peptide hydrogens exchange completely for deuterium at temperatures below those that lead to denaturation. Our results show that ProP adopts a highly alpha-helical fold similar to that of LacY, and that both transmembrane folds exhibit unusually high temperature-sensitive solvent accessibility. The results provide direct evidence that ProP adopts a structure consistent with other major facilitator superfamily members.