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FU Berlin
Digitale Dissertation

Christian Riebeling :
Regulationsmechanismen von Phospholipase D-Isoformen - Einfluß auf Differenzierung und Apoptose
Mechanisms of Regulation of Phospholipase D Isoforms - Involvement in Differentiation and Apoptosis

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Abstract

Dissertation Riebeling: Summary

4.1 Summary

The role and function of phospholipase D in cellular proliferation, differentiation and apoptosis is still not understood. Here, I describe the regulation of phospholipase D in some aspects of these cellular processes and the possible therapeutic intervention in one of these pathways.
The role of tyrosine phosphorylation in regulation of phospholipase D1 was investigated using a protein tyrosine phosphatase inhibitor. The induced accumulation of tyrosine phosphorylated proteins was accompanied by increased phospholipase D1 activity in vitro. Immunoprecipitation demonstrated that phospholipase D1 is not directly tyrosine phosphorylated in HaCaT keratinocytes in contrast to HL60 cells. These effects can be abolished using a protein tyrosine kinase inhibitor. As shown by Ras overexpressing HaCaT keratinocytes, the Ras/Raf/mitogen activated protein kinase pathway is not involved. This shows that tyrosine phosphorylation is a pathway indirectly regulating phospholipase D1.
Transcriptional regulation of phospholipase D in ceramide-induced apoptosis affects phospholipase D1. I could show that several differentiation-associated genes including phospholipase D1 are downregulated whereas members of the AP-1 transcription factor are upregulated. AP-1 is involved in the expression of differentiation-associated genes but the data presented suggest that the subunits which are upregulated in apoptosis form a repressor. In contrast, levels of phospholipase D2 mRNA are unchanged.
Moreover, I demonstrate that malignant melanoma exhibits augmented phospholipase D1 activity in comparison to primary cultured melanocytes. This increase is attained through enhanced protein expression in degenerated cells. In addition, although protein kinase Ca expression is not altered, activation of phospholipase D1 by phorbol ester is marginal in melanocytes in contrast to melanoma cells suggesting loss of an inhibitory factor of this interaction.
Cytoskeletal reorganisation is important for metastasis, and phosphatidic acid and the phospholipase D1 regulating Rho family proteins are involved in this process. Pamidronate interferes with Rho action via altering its membrane association. Here, I can show that pamidronate strongly induces apoptosis in melanoma cells. Addition of farnesol or geranylgeraniol reduces apoptosis by pamidronate with geranylgeraniol being a stronger inhibitor. This suggests geranylgeranylation being the more important step in pamidronate-induced apoptosis. However, one of the cell lines tested, Mel2A, is resistant against pamidronate mediated inhibition of proliferation and induction of apoptosis. This resistance is not achieved by alteration of the bax/bcl-2 ratio as was shown by bcl-2 overexpressing A375 cells. The levels of RhoB mRNA expression show no significant difference between melanoma cells and melanocytes. RhoA and RhoC in contrast are elevated in melanoma cells underscoring them as possible therapeutical targets. Although cytosolic RhoA levels are increased in all four melanoma cell lines after pamidronate treatment, levels of membrane-bound RhoA are not or only slightly altered. This suggests that decreased geranylgeranylation is counteracted by increased expression of Rho proteins. It remains to be investigated if the metastatic potential of these cells is nevertheless decreased.

Table of Contents

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Dissertation Riebeling: Contents

Contents

page
Title and contents
1. Introduction 1
1.1 Phospholipids in cellular signalling 1
1.2 Mechanisms of regulation of phosphatidylcholine-specific phospholipase D 3
1.3 Biochemistry of phosphatidylcholine-specific phospholipase D 14
1.3.1 Catalysis 14
1.3.2 Structure 16
1.4 Cellular functions of phosphatidylcholine-specific phospholipase D 18
1.4.1 Phosphatidic acid-derived diacylglycerol 19
1.4.2 Protein targets of phosphatidic acid 20
1.4.3 Vesicle transport 20
1.4.4 Stress fibre formation 21
1.5 Scope of this thesis 23
2. Results 24
2.1 Tyrosine phosphorylation of phospholipase D1 24
2.2 Expression of phospholipase D and differentiation-associated genes in ceramide induced apoptosis in HaCaT keratinocytes 27
2.3 Expression of phospholipase D and its stimulatory proteins in melanoma cells and primary cultured melanocytes 30
2.4 Effect of phorbol ester and guanosine-5'-O-(3-thiotriphosphate) on phospholipase D in melanoma cells 32
2.5 Effect of oleate on phospholipase D in melanoma cells 36
2.6 Effect of pamidronate on melanoma cells 37
3. Discussion 46
3.1 Effect of tyrosine phosphorylation on phospholipase D1 46
3.2 Expression of phospholipase D in ceramide-induced apoptosis 48
3.3 Regulation of phospholipase D in melanoma cells and melanocytes 50
3.4 Action of pamidronate on melanoma cells 52
4.1 Summary 55
4.2 Zusammenfassung 57
5. Materials 59
5.1 Reagents 59
5.2 Cell culture materials 60
5.3 Cell lines 60
5.4 Antibodies 61
5.5 Equipment 62
5.6 Primer 64
6. Methods 65
6.1 Cell culture 65
6.1.1 Growth media and solutions 65
6.1.2 Cultivation of cells 66
6.1.3 Freezing and thawing of cells 66
6.2 Lipid chemistry 67
6.2.1 Preparation of substrate vesicles for the in vitro phospholipase D assay 67
6.2.2 Identification and quantification of radioactively labelled lipids 67
6.3 Enzyme reactions 68
6.3.1 Assay of phospholipase D1 activity 68
6.4 Protein chemistry 69
6.4.1 Preparation of cell lysates and subcellular fractionation 69
6.4.2 Determination of protein concentration 69
6.4.3 Discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis 70
6.5 Immunochemistry 73
6.5.1 Purification of antibodies 73
6.5.1.1 Preparation of affinity columns 73
6.5.1.2 Affinity chromatography 74
6.5.2 Denaturing immunoprecipitation 75
6.5.3 Western blotting 76
6.5.4 Immunodetection of blotted proteins 77
6.5.5 Immunohistochemical detection using alkaline phosphatase anti alkaline phosphatase antibody (APAAP) complexes 79
6.6 Molecular biology 81
6.6.1 Isolation of total RNA 81
6.6.2 Determination of nucleic acid concentration 82
6.6.3 Synthesis of complementary DNA 82
6.6.4 Polymerase chain reaction 83
6.6.5 Agarose gel electrophoresis 84
6.7 Cell biology 85
6.7.1 Preparation of pervanadate 85
6.7.2 Measurement of proliferation 85
6.7.3 Determination of cytotoxicity 86
6.7.4 Detection of apoptosis 87
7. References 89
8. List of publications 108
8.1 Original publications 108
8.2 Short publications 108
8.3 Manuscripts 109
Curriculum vitae I
Acknowledgements II

More Information:

Online available: http://www.diss.fu-berlin.de/2001/100/indexe.html
Language of PhDThesis: english
Keywords: phospholipase D, phosphatidylcholine, ADP-ribosylation factor, RhoA, melanoma
DNB-Sachgruppe: 30 Chemie
Date of disputation: 25-Jun-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Dr. Christoph C. Geilen
Second Referee: Prof. Dr. Werner Reutter
Contact (Author): c_riebeling@yahoo.com
Contact (Advisor): ccgeilen@zedat.fu-berlin.de
Date created:20-Jun-2001
Date available:26-Jun-2001

 

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