Functional characterization of FMNL1 as potential target for novel anti-tumor therapies [Elektronische Ressource] / vorgelegt von Yanyan Han

Yanyan Han

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 44
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Aus dem Institut für Molekulare Immunologie
LeiterinPro f. Dolores Schendel
HMGU-München


Functional characterization of FMNL1
as potential target for novel anti-tumor therapies


Dissertation
Zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität München


vorgelegt von

Yanyan Han

aus Nanjing, China

2010

Mit Genehmigung der Medizinischen Fakultät
Der Universität München





Berichterstatter: Prof. Dr. Reinhard Zeidler

Mittberichterstatter: Prof. Dr. Christiane J. Bruns
Priv. Doz. Dr. Marion Subklewe


Mitbetreuung durch die
Promovierte Mitarbeiterin: Dr. med. Angela Krackhardt

Dekan: Prof. Dr. med, Dr. h.c. M.Reiser, FACR, FRCR

Tag der mündlichen Prüfung: 10.03.2010

Contents
Contents

1 Abstract .................................................................................. 1
2 Introduction ............................................................................ 5

2.1 Tumor antigens ..................................................................................... 5
2.1.1 Identification of tumor antigens ...................................................... 5
2.1.2 Characterization of tumor antigens ................................................ 7
2.1.3 Cancer immunotherapies based on tumor antigens ....................... 9

2.2 FMNL1 as a tumor associated antigens .............................................. 12
2.2.1 Identification of FMNL1 ................................................................ 12
2.2.2 Formin protein family .................................................................... 13
2.2.3 Diaphanous-related formins (DRFs) ............................................. 16

2.3 Non-Hodgkin's lymphoma (NHL) ........................................................ 18
2.3.1 Chronic lymphocytic leukemia (CLL) ........................................... 19
2.3.2 Therapies for CLL ........................................................................ 20

2.4 Aim of the project ................................................................................ 21

3 Materials................................................................................ 22

3.1 Equipments and Supplies .................................................................. 22

3.2 Chemicals, enzymes and cytokines ................................................... 23

3.3 Kits ...................................................................................................... 26

3.4 Buffers and solutions .......................................................................... 26

3.5 Cell culture medium ............................................................................ 28

3.6 Cell lines and bacteria ......................................................................... 29

3.7 Antibodies and peptides ...................................................................... 30

3.8 DNA-Material ...................................................................................... 31
3.8.1 Vectors ......................................................................................... 31
3.8.2 Primers ......................................................................................... 32 Contents
3.9 Software and website .......................................................................... 32

4 Methods................................................................................. 33

4.1 Cell culture method ............................................................................. 33
4.1.1 General cell culture methods ....................................................... 33
4.1.1.1 Freezing and thawing of cells .............................................. 33
4.1.1.2 Culture of cell lines ............................................................... 33
4.1.1.3 Determination of cell number ............................................... 33
4.1.1.4 Mycoplasma contamination test ........................................... 33
4.1.2 Isolation of PBL from whole blood ................................................ 34
4.1.3 Unspecific stimulation of T cells ................................................... 34
4.1.4 Peptide pulsing of T2 cells ........................................................... 34
4.1.5 Isolation of B cells from PBL using Magnetic Beads .................... 34
4.1.6 Unspecific Stimulation of B cells and CLL cells ............................ 35
4.1.7 Isolation and Maturation of Dendritic cells from PBL .................... 35

4.2 Protein biochemical methods .............................................................. 35
4.2.1 Western Blot ................................................................................. 35
4.2.1.1 Cell lysis and protein concentration mearsurment ............... 35
4.2.1.2 Electrophoresis and transfer ................................................ 36
4.2.1.3 Blotting ................................................................................. 36
34.2.2 [ H]- myristic acid uptake assay.................................................... 36
4.2.2.1 Immunoprecipitation ............................................................ 37
4.2.4.2 Autoradiography .................................................................. 37

4.3 Cell biological assays ......................................................................... 37
4.3.1 Immunofluorescence staining …………………………………….... 37
4.3.2 Apoptosis assay ........................................................................... 38
4.3.3 Proliferation Assay ....................................................................... 38
4.3.3.1 SNARF-1 staining ................................................................ 38
4.3.3.2 BrdU uptake assay .............................................................. 38
4.3.4 Analysis of free intracellular calcium concentration...................... 38

4.4 Protein overexpression ....................................................................... 39
4.4.1 Calcium-phosphate Transfection .................................................. 39
4.4.2 Adenoviral transduction ................................................................ 39
4.4.2.1 LR recombination reaction ................................................... 39
4.4.2.2 Generation and amplification of adenoviral stocks .............. 40
4.4.2.3 Titeration of adenoviral stocks ............................................. 40
4.4.2.4 Cell transduction with adenoviral stocks .............................. 40

4.5 Molecular biology method ................................................................... 41 Contents
4.5.1 Cloning of FMNL1  and FMNL1  into pcDNA 3.1 vector ............. 41
4.5.1.1 Amplification of 3` terminuses of FMNL1  and FMNL1   41 
4.5.1.2 Gel electrophoresis and gel-extraction of the PCR
production ........................................................................... 42
4.5.1.3 Digestion and purification of the inserts and vector ……….. 42
4.5.1.4 Ligation ................................................................................ 43
4.5.1.5 Transformation to bacteria ................................................... 44
4.5.1.6 Selection of the transformed bacteria .................................. 44
4.5.1.7 Plasmid-DNA-extraction and digestion ................................ 44
4.5.1.8 Plasmid sequencing and maxi-preparation .......................... 44
4.5.2 Cloning of Kozak sequence into pcDNA 3.1-FMNL1    44 
4.5.2.1 Amplification of 5` terminuse of FMNL1 containing kozak
sequence ............................................................................ 45
4.5.2.2 Digestion and purification of the inserts and vector ............. 46
4.5.2.3 Ligation ................................................................................ 46
4.5.2.4 Plasmid-DNA-extraction and digestion................................. 47
4.5.3 Cloning of FMNL1  /  into pACDC entry clone vector ................. 47 
4.5.3.1 Amplification of FMNL1    ………………………………….. 48
4.5.3.2 Digestion and purification of the inserts and vector ……….. 49
4.5.3.3 Ligation …………………………………………………………. 49
4.5.3.4 Plasmid-DNA-extraction and digestion ................................ 49
4.5.4 Mutagenesis ................................................................................... 50
4.5.4.1 Mutagenesis of N-termial myristoylation site in
pACDC-FMNL1  ................................................................. 50
4.5.4.2 Cloning of N-terminal myristoylation site mutant into pcDNA
3.1-FMNL1  ..................................................