Biomedical subjects

O Ammerpohl

Publications and source records attributed to O Ammerpohl.

10 recordsLinked to original sources

PubMed2006

Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.

Anthracenes↗

PubMed2006

Characterisation of the GRAF gene promoter and its methylation in patients with acute myeloid leukaemia and myelodysplastic syndrome.

We report the isolation of the 5' flanking region of GRAF (GTPase regulator associated with the focal adhesion kinase), previously described as a putative tumour suppressor gene of acute myelogenous leukaemia and myelodysplastic syndrome, and demonstrate its promoter activity in reporter gene assays. Two putative protein-binding sites are identified of which one was sensitive to CpG methylation. The suppressed GRAF expression could be restored in leukaemia cell lines by treatment with a demethylating agent and an inhibitor of histone deacetylases. In contrast to normal tissues, which tested negative for GRAF promoter methylation, 11 of 29 (38%) bone marrow samples from patients with acute myeloid leukaemia or myelodysplastic syndrome were positive.

Acute Disease↗

PubMed2004

ADAM9 expression in pancreatic cancer is associated with tumour type and is a prognostic factor in ductal adenocarcinoma.

Gene expression profiling revealed ADAM9 to be distinctly overexpressed in pancreatic ductal adenocarcinoma (PDAC). We examined the relevance of ADAM9 expression in PDAC diagnosis and prognosis. A total of 59 infiltrating PDACs, 32 specimens from patients with chronic pancreatitis, 11 endocrine tumours and 24 acinar cell carcinomas were immunohistochemically analysed for ADAM9 expression. Staining for ADAM9 was detected in 58 out of 59 (98.3%) PDACs and in two out of 24 (8.3%) acinar cell carcinomas, but not in endocrine tumours. In the non-neoplastic pancreas, whether normal or chronically inflamed, ADAM9 was expressed in centroacinar and intralobular duct cells, but not in interlobular duct cells and their hyperplastic lesions. Pancreatic ductal adenocarcinomas showing cytoplasmic ADAM9 expression correlated with poor tumour differentiation and also with shorter overall survival than in cases showing only an apical membranous staining pattern (P=0.001). Multivariate analysis identified cytoplasmic ADAM9 expression as an independent marker of shortened survival in a set of 42 curatively (R0) resected PDAC (P<0.05, hazard ratio 2.85, 95% confidence interval: 1.21-6.71). The results show that ADAM9 expression distinguishes PDACs from other solid pancreatic tumours. In addition, cytoplasmic ADAM9 overexpression is associated with poor differentiation and shortened survival. Therefore, ADAM9 overexpression might contribute to the aggressiveness of PDACs.

ADAM Proteins↗

PubMed2004

Histone deacetylase inhibitor 4-phenylbutyrate modulates glial fibrillary acidic protein and connexin 43 expression, and enhances gap-junction communication, in human glioblastoma cells.

Human glioblastoma cell cultures were established and the expression of glial fibrillary acidic protein (GFAP) and the gap-junction protein connexin 43 (Cx43) was confirmed by Western blot. Following treatment with 4-phenylbutyrate (4-PB), increased concentrations of non-phosphorylated GFAP were seen, while phosphorylated isoforms remained intact. Immunocytochemical staining of glioblastoma cells revealed an intracellular redistribution of GFAP. In addition to cytoplasmic immunostaining, GFAP immunoreactivity was also associated with the nucleus and/or the nuclear membrane. Phosphorylated and non-phosphorylated Cx43 proteins were increased 2- to 5-fold following 4-PB treatment, and were redistributed to areas of the cell surface, participating in cell-to-cell contacts. In addition, functional gap-junction coupling was amplified, as indicated by increased fluorescent dye transfer, and elevated levels of Cx43 protein were detected in parallel with enhanced gap-junction communication. Induced cell differentiation, with improved functional coupling of tumour cells, may be of importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds.

Antineoplastic Agents↗

PubMed2004

Histone deacetylase inhibitor 4-phenylbutyrate suppresses GAPDH mRNA expression in glioma cells.

The histone deacetylase (HDAC) inhibitor 4-phenylbutyrate (4-PB) is a non-toxic compound that can induce differentiation and promote maturation of various types of malignant cells. In the present study we show that 4-PB inhibit glioma cell proliferation, induce apoptosis and decrease mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in a concentration-dependent manner. Proliferation of established rat glioma cell lines (RG2 and C6) in culture was significantly decreased after treatment with 4-PB (2-40 mM). Low concentrations of 4-PB (2-20 mM) induced cell differentiation followed by apoptosis, whereas higher concentrations of 4-PB (40 mM) induced cell necrosis. Also, low concentrations of 4-PB significantly decreased GAPDH mRNA expression in C6 and RG2 rat glioma cells, suggesting a link between decreased cell proliferation, energy consumption, and down-regulation of GAPDH gene expression. We have found that GAPDH mRNA expression is markedly increased in human glioblastoma tissues. Therefore, the novel effect of 4-PB described here may offer means to suppress growth of glioma cells by diminishing the key reaction in glycolysis as a therapeutic approach for cancer.

Animals↗

PubMed2003

Characterization of lacZ complementation deletions using membrane receptor dimerization.

We describe the screening of lacZ deletions in mammalian cells and the discovery of a novel pair of lacZ deletions that can undergo alpha-complementation only when they are fused to peptides that interact with each other. The two lacZ deletions, delta N 11-75 and delta C 82-1023, were first characterized by fusing to two small interacting peptides and were then further analyzed by fusing to three membrane receptors (G protein-coupled receptors alpha 2cAR and D2DRL and receptor tyrosine kinase insulin receptor) that were known to form homodimers in the membrane. Histochemical and quantitative FACS assays demonstrated that the novel deletions have much lower level of association with each other, thus offering a much lower background in monitoring membrane protein interactions compared to previously published lacZ deletions. Furthermore, our method has the exciting potential to monitor simultaneously membrane receptor dimerization and localization to the cell surface of living cells.

Animals↗

PubMed2000

The minimal repression domain of MBD2b overlaps with the methyl-CpG-binding domain and binds directly to Sin3A.

Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.

Cell Line↗

PubMed1998

Repression of the mouse M-lysozyme gene involves both hindrance of enhancer factor binding to the methylated enhancer and histone deacetylation.

In many cases, gene repression mediated by CpG methylation has been demonstrated. Two different mechanisms have been postulated to explain the repressive effect of methylated CpG DNA: establishment of a repressive chromatin configuration and inhibition of DNA binding of transactivating factors. Using the M-lysozyme gene, we analyzed gene expression, CpG demethylation and the in vivo formation of enhancer/protein complexes after inducing demethylation or inhibiting histone deacetylases. We show that trans-cription of a methylated and silent mouse M-lysozyme gene can be induced upon the inhibition of histone deacetylases in the absence of demethylation or in vivo transactivating factor binding to the enhancer. In contrast, DNA demethylation induces both gene activity as well as enhancer complex formation. Therefore, both mechanisms play a role in lysozyme gene repression mediated by methylated DNA: (i) the enhancer cannot be loaded with transacting factors; and (ii) histone deacetylation inhibits transcription.

Acetylation↗

PubMed1997

Complex protein binding to the mouse M-lysozyme gene downstream enhancer involves single-stranded DNA binding.

The mouse M-lysozyme downstream enhancer has been previously characterized on several levels of gene regulation. The enhancer was co-localized with a DNase I hypersensitive site in the chromatin of mature macrophages, the in vivo interaction of transcription factor GABP with the enhancer core (MLDE) demonstrated binding being restricted to mature macrophage cells, and analysis of the MLDE methylation state revealed a correlation between demethylation of CpG dinucleotides and the in vivo GABP binding. Here, we analyzed in detail the full-length enhancer in addition to the core element. We identified a total of nine binding sites for nuclear factors. Most of these factors are found ubiquitously in all cell types tested. These factors include several unknown proteins as well as the transcription factor NF-Y. In addition, three binding sites for a new single-stranded DNA binding protein were found. The presence of this factor in mature macrophages correlates with the in vivo DNA melting of one of the binding sites and with the enhancer strength.

Animals↗

PubMed1997

Cis-elements required for the demethylation of the mouse M-lysozyme downstream enhancer.

The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.

Animals↗