Refine
Year of publication
- 2006 (2) (remove)
Document Type
- Doctoral Thesis (2) (remove)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Keywords
Institute
- Pharmazie (2)
Das Enzym 5-Lipoxygenase (5-LO) spielt eine essentielle Rolle in der Biosynthese der Leukotriene, bioaktiver Metabolite der Arachidonsäure (AA), die an einer Vielzahl entzündlicher und allergischer Erkrankungen beteiligt sind. Die 5-LO wird bevorzugt in Zellen myeloiden Ursprungs wie Granulozyten, Monozyten oder B-Lymphozyten exprimiert. In die Regulation der zellulären 5-LO-Aktivität in der Epstein-Barr Virus-transformierten B-lymphozytären Zelllinie BL41-E95-A sind Caspasen, Aspartat-spezifische Cysteinproteasen, involviert. Das Passagieren von BL41-E95-A führt zu einer Erhöhung der Proliferationsrate der B-Lymphozyten sowie zu einem deutlichen Verlust der 5-LO-Aktivität, der mit dem Auftreten eines 62 kDa-Spaltproduktes der 5-LO und einer signifikanten Aktivitätserhöhung der Caspase-8 und -6 korreliert. Isolierte humane 5-LO wird durch rekombinante Caspase-6 zwischen Asp170 und Ser171 zu einem 58 kDa-Fragment in vitro gespalten, wobei das Tetrapeptid VEID170 innerhalb der 5-LO als Erkennungsmotiv für den Angriff der Caspase-6 dient. In einigen weiteren untersuchten Zelllinien wie Mono Mac 6 (MM6), RBL-1, PMNL oder HeLa, die nicht den B-Lymphozyten angehören, konnte die 5-LO-Spaltung weder durch das Passagieren von Zellen noch durch die Behandlung mit diversen proapoptotischen Agentien ausgelöst werden. Laut Ergebnissen aus in vitro-Untersuchungen scheinen 5-LO-positive HeLa- bzw. MM6-Zellen einen Faktor zu exprimieren, der die 5-LO direkt oder indirekt vor dem Angriff der Caspase-6 und anschließender Prozessierung schützt. Die in den BL41-E95-A-Zellen beobachtete Aktivierung der Caspasen mit anschließender Prozessierung der 5-LO lässt sich durch zwei Pflanzeninhaltsstoffe supprimieren, das Hyperforin (HP) aus Johanniskraut-Extrakten und das Myrtucommulon (MC) aus Myrte-Blättern. Beide Verbindungen scheinen in B-Lymphozyten zu einer Hemmung der Caspasen-Aktivierung zu führen. Nichtsdestotrotz führt die Behandlung der B-Lymphozyten mit HP bzw. MC zu einem apoptotischen Tod der Zellen. Offensichtlich wird dabei ein (unbekannter) einzigartiger Mechanismus der Apoptose-Induktion ausgelöst. In der vorliegenden Arbeit konnte zum ersten Mal eine potente Apoptose-induzierende Wirkung des natürlich vorkommenden Myrtucommulons auf Krebszelllinien gezeigt werden. In allen getesteten Krebszelllinien führte Myrtucommulon zum Zelltod, wobei die HL-60-Zellen mit einem IC50-Wert von 3,26 ± 0,51 µM MC am sensitivsten gegenüber MC-Einfluss waren. Zusätzlich konnte in HL-60- und MM6-Zellen nach MC-Behandlung neben einer erhöhten Caspasen-Aktivität und PARP-Spaltung ein signifikanter DNA-Abbau detektiert werden. Von besonderer Bedeutung ist die Tatsache, dass die zytotoxische MC-Wirkung eine bemerkenswerte Selektivität für entartete Zelllinien zu besitzen scheint und gegenüber nicht-transfizierten Zellen minimal ist.
5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes, converting arachidonic acid to 5-HPETE, and in a second step 5-HPETE to leukotriene A4. Although the 5-LO promoter possesses characteristics of so called housekeeping genes, such as lack of TATA/CCAAT boxes and existence of several Sp1 binding sites, the 5 -LO gene is tissue specifically expressed in primarily immune competent cells of myeloid origin including granulocytes, monocytes, macrophages, mast cells and B-lymphocytes. 5-LO gene expression in MM6 and HL-60 cells is strongly induced after differentiation of the cells with TGF-beta and 1,25(OH)2D3. In some monocytic cancer cell lines, such as HL-60 TB and U937, TGF-beta and 1,25(OH)2D3 treatment are not able to activate 5-LO gene transcription. It was demonstrated, that in these cell lines the 5-LO core promoter is heavily methylated and that only demethylation by the DNA methyltransferase inhibitor 5-aza-2 deoxycytidine (Adc) upregulated the 5-LO mRNA levels. It was also shown that the histone deacetylase inhibitor TsA could induce 5-LO mRNA levels, but only in 1,25(OH)2D3/TGF-beta inducible MM6 cells. Interestingly the 1,25(OH)2D3/TGF-beta effect on 5-LO expression is reduced, when combined with TsA. Reporter gene assays revealed that 5-LO promoter activity is strongly induced after 24 h treatment with 330 nM TsA (construct N10 up to 35 fold in HeLa cells). The effect is dependent on the presence of the proximal Sp1 binding site GC4 (-53 bp to –48 bp in relation to the major TIS) in both HeLa and MM6 cells. In vitro binding of the transcription factor Sp1 to this site has been demonstrated in gel shift assays and DNase I footprints. Mutation of the binding site resulted in a loss of basal promoter activity in both 5-LO negative HeLa cells and in 5-LO positive MM6 cells, as well as in the loss of TsA inducibility. The mutational study of different Sp1 binding sites in a larger promoter context revealed the interaction or respectively the additive effect of the multiple Sp1 binding sites of the 5-LO promoter on basal as well as on TsA upregulated promoter activity. However, GC4 seems to be of special relevance for both the basal promoter activity, possibly recruiting the basal transcription machinery, as well as for the TsA induced upregulation of 5-LO promoter activity. TsA does not alter the protein expression levels of Sp1 and Sp3 as investigated in Western blot analysis, neither in HeLa nor in MM6 cells. DNA affinity purification assays revealed that TsA had no effect on the DNA affinity of Sp1 or Sp3. In vitro binding of both Sp1 and Sp3 to the 5-fold GC box, GC4 and GC5 was demonstrated by DAPA analysis, but histone deacetylase inhibition did not change the associated protein amounts. Finally, in vivo binding of Sp1 and Sp3 was investigated in chromatin immunoprecipitation assay (ChIP) in MM6 cells. TsA clearly induced the association of both proteins to the promoter area surrounding the TIS. Upon TsA treatment also RNA polymerase II binding to the area surrounding the TIS (-318 to +52 bp) was increased and even initiated in the more distal promoter parts –1049 to –292 bp, which are negatively regulated in reporter gene assays. Interestingly histone H4 is already highly acetylated without TsA treatment and the acetylation status of H4 remains unchanged after histone deacetylase inhibition, indicating an open chromatin structure of the 5-LO gene in MM6 cells. In a cotransfection study with Sp1 and Sp3, the transactivating potential of factors was investigated and in accordance with the ChIP data, Sp1 and Sp3 increased the promoter activity, but only after TsA treatment. In gel shift assays, the influence of DNA methylation on Sp1 binding was investigated. The results indicate different roles for the three proximal promoter sites. Whereas Sp1 binding to the 5-fold GC box and GC4 is impaired by DNA methylation, binding to GC5 is even increased. A cotransfection study with methylated 5-LO promoter constructs and the murine methyl-CpG binding proteins suggest MBD1 involvement in the regulation of the 5-LO promoter. Since in gel shifts Sp1 binding is inhibited by DNA methylation, at least to the 5-fold GC box and the activating element GC4, and similarly the mutation/deletion of the same sites strongly reduces or inhibits promoter activity, it is likely to assume, that the loss of promoter activity after in vitro methylation is in the first place due to impaired Sp1/Sp3 binding. Together the data underline the importance and complexity of Sp1/Sp3 binding to the GC rich sites in the regulation of 5-LO promoter activity in response to the histone deacetylase inhibitor TsA as well as in respect to DNA methylation.