Establishment and application of a highly sensitive coupled luminescent method (CLM) to study natural killer cell cytolytic activity [Elektronische Ressource] / von Henry Ogbomo

Viro-Research 05

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• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Establishment and application of a highly sensitive
coupled luminescent method (CLM) to study natural
killer cell cytolytic activity



Dissertation
Zur Erlangung des Doktorgrades
der Naturwissenschaften




vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main



von
Henry Ogbomo
aus Benin City, Nigeria



Frankfurt (2008)

(D30)









vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang Goethe-
Universität als Dissertation angenommen.









Dekan : Prof. Dr. Harald Schwalbe

Gutachter : Prof. Dr. Jörg Kreuter; Prof. Dr. Jindrich Cinatl

Datum der Disputation: 04.06.2008







Dedicated to my familyTABLE OF CONTENT

1.0 Introduction.........................................................................................................................1

1.1 Regulation of NK cell function.............................................................................................2

1.2 NK cell inhibitory receptors………………………………………………………………..3

1.3 NK cell activating receptors………………………………………………………………..5

1.3.1 Natural cytotoxicity receptors (NCRs)..…………………………………............………7

1.3.2 Natural killer group 2D (NKG2D)……………………............………………………….7

1.3.3 NK cell antibody receptors…………...........………………………………………….....9

1.3.4 NK cell co-stimulatory molecules………...........………………………………………10

1.3.5 Cytokine and chemokine regulation of NK cells…………............……….……………11

1.4 Biology of histone deacetylases (HDACs) and histone acetyltransferases (HAT)…….…11

1.5 HDAC substrates……………….…………………………………………………………13

1.6 HDACs and HATs in cancer….…………………………………………………………..13

1.7 Histone deacetylase inhibitors (HDACi)…………………………………………………14

1.8 Biologic activities of HDACi………………………………………………………….….17

1.8.1 HDACi and cell cycle arrest……………...........…………………………………….…17

1.8.2 Apoptotic effects of HDACi………………………………………............……………18

1.8.3 Antiangiogenic effects of HDACi……………………………………...........…….…....19

1.8.4 HDACi effects in animal studies……………………………………..........…….….….19

1.9 Clinical trials with HDACi……………………………………………………………….20

1.9.1 SAHA (Vorinostat)…………………………………………………............…………..21

1.9.2 VPA…………………………………………………............…………………………..22

1.10 HDACi and the immune system………………………………………………………...23

1.11 Nucleoside analogs……………………………………………………………………...25

1.12 1- β-D-arabinofuranosylcytosine (cytosine arabinoside, cytarabine, araC)…………..….25

1.13 Mechanisms of resistance to araC in transformed cells……………………………..…..26 1.14 Methods for measuring NK cell cytotoxicity....................................................................27

1.15 Aim of research………………………………………………………………………….29


2.0 Materials and Methods………………………………………….………………………31

2.1 Materials…………………………………………………………………………………..31

2.1.1 Chemicals………………………………………………………………............……….31

2.1.2 Media, buffer and solution…………………………………………………............…...34

2.1.2.1 Media, buffer and cell culture solution……………………………….........................34

2.1.2.2 Buffer and solution for fluorescent-activated cell sorting (FACS)…...........................36

2.1.2.3 Buffer for cell separation (MACS buffer)………………………........................….....36

2.1.2.4 Buffer for cDNA (RTA buffer)…………………………………….............................36

2.1.3 Antibodies and cytokines………………………………………………….............……36

2.1.3.1 Monoclonal antibodies……………………………………………........................…..36

2.1.3.2 Antibodies for western blot……………………………………........................……...39

2.1.3.3 Cytokines………………………………………………………….......................…...40

2.1.4 Reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE)……….................40

2.1.4.1 Resolving gel composition…………………………………………........................…40

2.1.4.2 Stacking gel co……………………………………........................……..40

2.1.4.3 Protease inhibitor mix………………………………………….......................……....41

2.1.4.4 Electrophoresis and transfer buffer…………………………….......................……....41

2.1.4.5 Wash and blocking buffers…………………………………….......................………41

2.1.5 PCR……………………………………………………………………...........…….…..42

2.1.5.1 Reagents for reverse transcription polymerase chain reaction……........................…..42

2.1.5.2 Primers………………………………………………………….......................….......43

2.1.6 Commercial kits…………………………………………………………...........….…...43

2.1.7 Diverse materials…………………………………………………………...........…..….44
2.1.8 Laboratory equipments and software……………………………………...........….…...45

2.1.9 Target cell lines……………………………………………………………............……48

2.1.9.1 Cell lines purchased from cell bank…………………………………..........................48

2.1.9.2 Established cell lines……………………………………………........................…….50

2.2 Methods…………………………………………………………………………………...51

2.2.1 Cultivation of adherent eukaryotic cells………………………………………..............51

2.2.2 Cultivation of eukaryotic suspension cells…………………………………............…...51

2.2.3 Polyclonal NK cell preparation……………………………………………....................52

2.2.4 Cytotoxicity assay principle……………………………………….……............………53

2.2.4.1 Redirected lysis.............................................................................................................54

2.2.5 The flow cytometric principle……………………………………….……............…….55

2.2.5.1 Determination of cell cycle using propidium iodode………….……...........................59

2.2.5.2 Effect of HDACi on NK cell viability………………………….......................….…..60

2.2.5.3 Measurement of cell surface receptors……………….…………….......................…..60

2.2.6 Principle of RT-PCR……………………………………………………............…........61

2.2.7 Real-time RT-PCR (SYBR green principle)………………………………....................63

2.2.7.1 Real.time RT-PCR………………………………………………................................63

2.2.8 NK receptor cross-linking and perforin/granzyme B granule release……..............……64

2.2.9 Measurement of IFN- γ production……………………………………............…..…….65

2.3.0 Measurement of NF κB activation………………………………………..............……..65

2.3.1 MTT assay………………………………………………………………...............…….65

2.3.1.1 Cytotoxicity of araC on leukemic cells…………………………….............................66

2.3.2 Western blot principle…………………………………………………............………..67

2.3.2.1 Western blot analyses of leukemic cells…………………………….......................…68


3.0 Results…………………………………………………………………………………....70
3.1 Purity of NK cells...............................................................................................................70

3.2 Establishing the coupled luminescent method (CLM) for measuring cytotoxicity………70

3.3 Validating CLM using NB as target cells...........................................................................72

3.4 Validating CLM using other cell types as target………………………………………....74

3.5 Effect of HDACi on viability of NK cells…………………………………………..……75

3.6 HDACi suppress IL-2-mediated NK cell cytotoxicity…………………………..………..76

3.7 HDACi down-modulate NK cell activating receptors expression……………………..…78

3.8 HDACi suppress NK cell function……………………………………………………..…81

3.9 HDACi impair granule exocytosis and inhibit IFN- γ production…………………….…..82

3.10 SAHA and VPA suppress NF κB activation in IL-2-activated NK cells………………...84

3.11 Viability of leukemic cells upon araC treatment…………………………………….…..86

3.12 Cytotoxic activity of IL-2-activated NK cells against leukemic cell lines……………...86

3.13 Expression of NK cell activating and inhibitory ligands in leukemic cells…………..…87

3.14 NK cell recognition of leukemic cell lines via NKG2D………………………………...89

3.15 Possible mechanism of increased ligand expression in araC-resistant leukemic cells….90

3.16 Role of ERK signaling in NKG2D ligand expression…………………………………..92


4.0 Discussion…………………………………………………………………………….…..94

4.1 Luminescent assay………………………………………………………………………..94

4.2 Establishing CLM to measure NK cell cytotoxicity……………………………………...95

4.3 SAHA and VPA suppress IL-2-mediated NK cell cytotoxicity………………………….96

4.4 HDACi-treated NK cells repress HDACi-induced enhanced NK cell sensitivity of
leukemic cells………………………………………………………………………………...98

4.5 Mechanism of HDACi inhibition of NK cell cytolytic activity………………………….98

4.6 AraC-induced resistance of leukemic cells increases their sensitivity to NK cell lysis…..99

4.7 Mechanism of increased sensitivity of araC-resistant leukemic cells…………………...100