DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Gerit Goltz :
Characterization of ceramidase inhibitors in the human keratinocyte cell line HaCaT
Charakterisierung von Ceramidase-Inhibitoren an der humanen Keratinozyten-Zellinie HaCaT

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Abstract


In the present dissertation the biological effects of phenylaminoalcohol analogues D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-e-MAPP), L-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (L-e-MAPP) and the newly synthesized analogue D-threo-2-(N-myristoylamino)-1-(4-nitrophenyl)-1,3-propandiol (D-NMAPPD or B13) were studied in HaCaT keratinocytes.
In recent publications D-e-MAPP was shown to inhibit the alkaline ceramidase leading to increased level of intracellular ceramide and inhibition of cell growth in HL-60 cells. In contrast, we could demonstrate in HaCaT keratinocytes that D-e-MAPP and B13 inhibit the acid ceramidase activity whereas B13 was the most potent inhibitor. All three derivatives did not influence the activity of the alkaline ceramidase. L-e-MAPP on the other hand was an inactive analogue.
Ceramidase inhibition was followed by time and concentration dependent increase of intracellular ceramide level with subsequent increase of apoptosis and suppression of proliferation of HaCaT keratinocytes. B13 was again more potent in inducing apoptosis and inhibiting cell growth.
Bcl-2 overexpression abolished apoptosis triggered by B13 indicating that the generated ceramide leads to apoptosis via mitochondrial cytochrome c-release and following caspase activation.
In addition to stereospecificity -only D-stereoisomers were active- other structural features might be important for substrate recognition and binding of ceramidase and thereby affecting its biological function.
We could demonstrate in HaCaT keratinocytes that the endogenous ceramide level can be modulated by influencing the acid ceramidase activity followed by ceramide-specific biological responses. These findings show that acid ceramidase is not only involved in lysosomal degradation of sphingolipids as previously described but is a key enzyme in ceramide metabolism and cellular signal transduction. The investigated ceramidase inhibitors might represent a new class of drugs in the therapy of benign hyperproliferative skin diseases such as psoriasis vulgaris as well as in malignant skin diseases.

Table of Contents

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0. Titelblatt und Inhaltsverzeichnis
1. Einleitung 1
     
1.1 Struktur und Biosynthese von Ceramid 1
1.2 Der Sphingomyelinzyklus 2
1.3 Biologische Rolle von Ceramid 4
1.3.1 Ceramid und Apoptose 4
1.3.2 Haut und Apoptose 9
1.3.3 Rolle von Ceramid in der Haut 10
1.4 Ceramid-vermittelte Signalwege 11
1.5 Modulation intrazellulärer Ceramid-Spiegel 14
1.5.1 Erhoehung des Ceramid-Spiegels 14
1.5.2 Verminderung des Ceramid-Spiegels 16
1.6 Ceramidasen 18
1.6.1 Vorkommen und Isoformen der Ceramidase 18
1.6.2 Ceramidase-Inhibitoren 21
1.7 Fragestellung der Arbeit 22
     
2. Material 24
     
2.1 Geraete 24
2.2 Zellinien 25
2.3 Phenylaminoalkohol-Derivate 25
2.4 Radioaktivität 26
2.5 PCR-Reagenzien 26
2.6 Chemikalien 27
2.7 Zellkulturmaterialien 27
     
3. Methoden 29
     
3.1 Zellkultur 29
3.1.1 Zellkulturmedien 29
3.1.2 Kultivierung der Zellen 30
3.1.3 Einfrieren und Auftauen der Zellen 30
3.2 Behandlung der Zellen mit den Phenylaminoalkohol-Derivaten 31
3.3 Messung der Zytotoxizität 31
3.4 Messung der Apoptose 33
3.5 Messung der Proliferation 35
3.6 Proteinbestimmung mit Bicinchoninsäure 37
3.7 Lipidchemische Methoden 39
3.7.1 Lipid-Extraktion 39
3.7.2 Duennschichtchromatographische Trennung von Lipiden 40
3.7.3 Nachweis radioaktiv-markierter Lipide 40
3.7.4 Nachweis nichtradioaktiver Lipide 41
3.8 Bestimmung der Ceramidase-Enzymaktivität 41
3.9 Messung des Ceramid-Gehaltes 44
3.10 Polymerase-Kettenreaktion 45
3.11 Statistische Auswertung der Ergebnisse 48
     
4. Ergebnisse 49
     
4.1 Bestimmung der Zytotoxizität der Phenylaminoalkohol-Derivate L-e-MAPP, D-e-MAPP und B13 auf HaCaT-Keratinozyten 49
4.2 Apoptotischer Effekt der Phenylaminoalkohol-Derivate L-e-MAPP, D-e-MAPP und B13 auf HaCaT-Keratinozyten 52
4.3 Einfluß von bcl-2 auf die Phenylaminoalkohol-induzierte Apoptose 55
4.4 Beeinflussung des Wachstumsverhaltens von HaCaT-Keratinozyten durch die Phenylaminoalkohol-Derivate L-e-MAPP, D-e-MAPP und B13 56
4.5 Darstellung der sauren Ceramidase-Isoform in HaCaT-Keratinozyten mittels RT-PCR 59
4.6 Effekte der Phenylaminoalkohol-Derivate auf die Ceramidase-Isoformen in HaCaT-Keratinozyten 60
4.7 Beeinflussung des intrazellulären Ceramid-Spiegels durch die Phenylaminoalkohol-Derivate L-e-MAPP, D-e-MAPP und B13 63
     
5. Diskussion 65
     
6. Zusammenfassung 76
     
7. Literaturverzeichnis 78
     
8. Veroeffentlichungen 91
     
Danksagung I
     
Lebenslauf II

More Information:

Online available: http://www.diss.fu-berlin.de/2002/180/indexe.html
Language of PhDThesis: german
Keywords: apoptosis, ceramidase, b13, d-e-mapp, bcl-2
DNB-Sachgruppe: 33 Medizin
Date of disputation: 09-Jul-2002
PhDThesis from: Fachbereich Humanmedizin, Freie Universität Berlin
First Referee: Prof. Dr. Dr. Christoph C. Geilen
Second Referee: Prof. Dr. Werner Reutter
Contact (Author): geritgoltz2002@yahoo.com
Contact (Advisor): ccgeilen@zedat.fu-berlin.de
Date created:04-Sep-2002
Date available:10-Sep-2002

 

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