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A New Role for Neurotransmitters: Inhibitors of Cell Migration

Inaugural-Dissertation

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

an der

Fakultät für Biowissenschaften

der Universität Witten/Herdecke

vorgelegt von:

LmCh. Jan Joseph

aus Prüm

Witten im Sommer 2004

II

Mentor (Erstgutachter): Herr Prof. Dr. Dr. Kurt S. Zänker

Fakultätsreferent (Zweitgutachter): Herr Prof. Dr. H. –P. Bertram

Externer Referent:

Tag der Disputation: 04. Oktober 2004

III

If chemistry fits, the body could fight against cancer and metastasis on it`s own.This fight could be made bearable with the use of novel pharmacological drugs.

Ich schlief und träumte, das Leben sei Freude; ich erwachte und sah das Leben war Pflicht:

ich handelte und siehe: die Pflicht ward Freude.

Rabindranath Tagore

Contents IV

Contents

ABBREVIATIONS VI

ZUSAMMENFASSUNG VIII

SUMMARY X

INTRODUCTION 1

CANCER 1

METASTASIS DEVELOPMENT 2

IMMUNE SYSTEM 4

CELL MIGRATION 9

THE INITIAL STEP FOR MIGRATION – STEP ZERO 10

KEY REGULATORS OF THE SIGNAL TRANSDUCTION FOR MIGRATION 12

INDUCERS FOR MIGRATION 13

NEUROTRANSMITTERS WITH INHIBITORY FUNCTION 15

GABA 15

ANANDAMIDE 17

G PROTEIN-COUPLED RECEPTORS 19

THE AIMS OF THIS STUDY 22

EXPERIMENTAL PROCEDURES 23

MATERIAL 23

TABLE OF PHARMACOLOGICAL SUBSTANCES USED IN THIS WORK 23

EQUIPMENT 25

CELL CULTIVATION AND ISOLATION 26

TUMOUR CELL LINES 26

T LYMPHOCYTES 26

NEUTROPHIL GRANULOCYTES 27

Contents V

CELL MIGRATION ASSAY 28

EGFP-ACTIN VECTOR CONSTRUCTION 31

TRANSFORMATION PROTOCOL 32

ISOLATION OF PLASMID DNA 32

THE CONTROL OF THE DNA PREPARATION WITH RESTRICTION ENZYMES 34

TRANSFECTION OF SW 480 COLON CARCINOMA CELLS 35

CONFOCAL LASER SCANNING MICROSCOPY 36

FLOW-CYTOMETRICAL MEASUREMENT OF CYTOSOLIC CALCIUM 37

FLOW-CYTOMETRICAL DETECTION OF CANNABINOID-RECEPTORS 37

MEASUREMENT OF CELLULAR CAMP 38

IMMUNOBLOTTING OF PROTEIN TYROSINE PHOSPHORYLATION 38

RESULTS 40

CELL MIGRATION REGULATED BY GABA 40

EFFECT OF GABA ON THE MIGRATION OF TUMOUR CELLS 40

EFFECT OF GABA ON THE MIGRATION OF LEUKOCYTES 48

CELL MIGRATION REGULATED BY ANANDAMIDE 50

EFFECT OF ANANDAMIDE ON THE MIGRATION OF TUMOUR CELLS 50

EFFECT OF ANANDAMIDE ON THE MIGRATION OF LEUKOCYTES 53

DISCUSSION 56

ACKNOWLEDGEMENTS 64

REFERENCES 65

CURRICULUM VITAE 72

PUBLICATIONS 73

Abbrevations VI

Abbreviations

2-AG 2-arachidonoyl glycerol

Ab/mAb antibody/monoclonal antibody

AA amino acids

AC adenylyl cyclase

APCs / pAPCs antigen presenting cells / professional APCs

cAMP cyclic adenosin-monophosphate

CB1/2–R cannabinoid 1/2 receptor

CD cluster of differentiation

CTX cholera toxin

DAG diacylglycerol

DAO diamine oxidase

DC dendritic cell

DEA docosatetraenamide

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

EGF epidermal growth factor

EGFP enhanced green fluorescent protein

FAK focal adhesion kinase

FAB fragment antigen binding

FCS fetal calf serum

FITC fluorescein isothiocyanate

fMLP formyl-methionyl-leucyl-phenylalanine

Abbrevations VII

GABA γ-aminobutyric acid

GAD glutamate decarboxilase

GBR1/2 GABAB1/2 receptor

GPCRs guanine-nucleotide binding protein coupled receptors

IL interleukin

IP3 inositol-1,4,5-phosphate

MARCKS myristoylated, alanine-rich C kinase substrate

MFI mean fluorescence intensity

MHC major histocompatibility complex

NK cells natural killer cells

PBS phosphate-buffered saline

PDGF platelet-derived growth factor

PEA palmitoyl ethanolamide

PIP2 phosphatidylinositol-4,5-phosphate

PKA/C protein kinase A/C

PLB phospholamban

PLCβ/γ phospholipase C β/γ

PMA phorbol-12-myristate-13-acetate

PTK protein tyrosine kinase

PTP protein tyrosine phosphatase

PTX pertussis toxin

ROCK rho-associated coiled-coil forming kinase

RTK receptor tyrosine kinase

SDF-1 stromal cell-derived factor-1

SERCA sarcoplasmatic/endoplasmatic reticulum calcium ATPase

SSADH succinic semialdehyde dehydrogen

Zusammenfassung VIII

Zusammenfassung

Die Wanderung von Zellen ist eine wesentliche Voraussetzung für die

Embryogenese. Embryonale Stammzellen eines sich entwickelnden

Organismus wandern zu ihrer vorbestimmten Position und differenzieren dort.

Im adulten Organismus wandern nur wenige Zellen, wie beispielsweise die

Zellen des Immunsystems. Lymphozyten und neutrophile Granulozyten

migrieren, um den Ort einer Infektion zu erreichen und dort entsprechende

Aufgaben der Immunantwort zu erfüllen. Der physiologischen Migration dieser

Leukozyten steht die pathologische Migration von Tumorzellen gegenüber.

Diese Wanderung von Tumorzellen ist die Voraussetzung für die Entwicklung

von Metastasen.

Chemokine und Zytokine sind bekannte Regulatoren für die Wanderung von

Zellen. Wir haben nun beobachtet, dass Neurotransmitter ebenso die Migration

von Zellen regulieren können. Die vorliegende Arbeit befasst sich mit zweien

dieser Neurotransmitter, γ-Amino-Buttersäure (GABA) und Anandamide, welche

als Inhibitoren für die noradrenalininduzierte Wanderung von Zellen des Kolon-

und Mammakarzinom fungieren und ebenso die SDF-1-induzierte Wanderung

von CD8+ T-Lymphozyten hemmen können. Die Signaltransduktion dieser

Inhibition der Wanderung von Tumorzellen wurde mit der von Zellen des

Immunsystems verglichen, um mögliche Unterschiede erkennen zu können.

Durch Einbetten der Zellen in eine dreidimensionale Kollagenmatrix und video-

mikroskopische Zeitraffer-Aufnahmen wurde die Wanderungsaktivität der Zellen

bestimmt, und durch pharmakologische Inhibitoren und Aktivatoren einzelner

Zusammenfassung IX

Enzyme sowie molekularbiologische Methoden konnten Rückschlüsse auf die

zugrunde liegende Signaltransduktion gezogen werden.

Durch rezeptorspezifische Agonisten wurde gezeigt, dass die Regulation der

Zellmigration durch GABA und Anandamide über Gi/Gs-Protein gekoppelte

Rezeptoren beeinflusst wird. Die initiierten Pfade der Signaltransduktion sind im

wesentlichen durch zwei Elemente bestimmt: zum Einen ist dies die Regulation

von cAMP, zum Anderen der Ein- und Ausstrom von Kalzium aus intrazellulären

Speichern. Da der Effekt von GABA und Anandamide in Tumorzellen über

andere Rezeptoren vermittelt wird als in T-Lymphozyten, könnten der

spezifische GABAB-Rezeptor Agonist Baclofen, wie auch der spezifische CB1-

Rezeptor Agonist DEA potentielle pharmazeutische Inhibitoren für die Migration

von Tumorzellen sein, und somit potentielle Wirkstoffe gegen die Entwicklung

von Metastasen darstellen. Mit dem weiteren Wissen der

Signaltransduktionwege könnte es möglich sein, die Wanderung der

Tumorzellen selektiv zu hemmen ohne das Immunsystem zu beeinflussen.

Summary X

Summary

The migration of cells is essential in embryogenesis, where the differenting cells

of an organism migrate to their appropriate position. In an adult organism only a

few specialized cells migrate. For example, cells of the immune system like

lymphocytes and neutrophil granulocytes migrate in order to reach sites of

infection. Tumour cells are able to migrate, too, which is a prerequisite for the

development of metastases.

It is known that chemokines and cytokines are regulators for migration. Here we

have observed that neurotransmitters are migration-regulators, too. We have

focussed on the neurotransmitters, γ-aminobutyric acid (GABA) and

arachidonoylethanolamide (anandamide), which are inhibitors for the

norepinephrine induced migration of colon carcinoma cells and breast cancer

cells and the SDF-1 induced migration of CD8+ T lymphocytes. We have

investigated the signal transduction of migrating cells of the immune system in

comparison to the migration of tumour cells. To investigate the signal

transduction of migration we incorporated the cells into a three-dimensional

collagen matrix and recorded their locomotor behaviour by time-lapse

videomicroscopy. We used molecular-biological and pharmacological methods

to specifically interfere with certain steps of the signal transduction pathways. In

combination with other biochemical methods we elucidated different pathways

of cell migration.

By starting the investigation of the signal transduction with receptor specific

agonists, we found that Gi/Gs protein-coupled receptors (GPCRs) are involved

Summary XI

in these processes. The initiated signal transduction pathways were integrated

on two regulatory events: one is the regulation of cAMP, and the second is the

influx of calcium from and the sequestration of calcium into intracellular stores.

The specific GABAB-receptor agonist baclofen as well as DEA as a specific

CB1-receptor agonist are potentional pharmaceutical migration inhibitors and

might be potential tools against metastases development. With the further

knowledge of the signal transduction pathways it might be possible to

selectively inhibit the migration of tumour cells without interfering with the

migration of immune cells.

Introduction 1

Introduction

Cancer

In the period from 1999 to 2002 in Germany more than 210,000 people died per

year from cancer or so-called neoplasm. To die from a neoplasm is the second

highest cause of death, after dying from circulation diseases, with more than

390,000 causes of death [Statistisches Bundesamt D, 2004].

Proliferation, differentiation, apoptosis and migration are normal functions of

cells. Irreversible changes in the structure or in the expression of genes caused

by chemical carcinogenic substances, physical carcinogens (radiation) or

tumourigenic viruses can destroy the balance of proliferation, differentiation and

apoptosis of cells. These resulting genetic alterations can initiate

carcinogenesis [Schmoll H J, 1999]. The three main exogenous factors for

cancer development include diet, occupation and pollution [Tomatis L, et al.,

1997]. The main established risk factors for cancer are (i) smoking, which

accounts for over 30% of all cancer diseases, especially of cancer localised in

lung, mouth, throat, oesophagus and cervix, (ii) alcohol consumption for cancer

localised in mouth, throat and oesophagus, (iii) obesity for tumours in the

uterine. These are three examples of risk factors but there are protective

factors, too. A diet of fruit and vegetables is protective against cancer in mouth,

throat, oesophagus and stomach. A diet of vegetables or exercise could be

protective factors for colon cancer development [Glade M J, 1999].

Introduction 2

The three most common kinds of cancer in Germany are lung cancer (4.6%),

colon cancer (2.4%) and breast cancer (2.1%) (Percentages of causes of death,

Germany 2001) [Statistisches Bundesamt D, 2004].

Metastasis development

The migration of tumour cells is a prerequisite for the development of

metastases. The growth of benign tumour cells differs from the growth of

malignant tumour cells in the aspect, that it has no competence for metastases

development. Benign growing furthermore does not infiltrate normal tissue.

The first step of metastasis development is an expansive proliferation of the

primary tumour [Fidler I J, Poste G, 1982]. A subpopulation of tumour cells

migrates into vascular or lymphatic structures and is disseminated by the blood

or lymph stream. The circulating tumour cells adhere to the vessels at distant

organs and invade into the subendothelial tissue. The metastatic cells start to

proliferate again and the metastasis is apparent (Fig. 1).

Introduction 3

Figure 1: Sequential “metastasis cascade”. The primary tumour (black field) proliferates(step 1.). Invasion of primary tumour cells in lymphatic or capillary vessels (step 2.). Formationof tumour-emboli (step 3). Adhesion of tumour cell(s) to membrane vessels (step 4.). Invasion oftumour cells into subendothelial areas (step 5.). The metastatic cells start to proliferate again(step 6.).

The localisation of metastasis development is not random. The distribution of

metastases shows a pattern independent of the rate of blood flow [Glade M J,

1999]. Organs with a high blood volume throughput like heart, muscles, kidney,

gut and spleen have a relatively low risk for the occurrence of metastases

[Nicolson G L, 1988]. An explanation for the controversly discussed non-random

occurrence is given by the “Seed-and-soil-theory” of Paget 1889: in this

explanation the tumour cell (seed) come across a fertile target (soil) to

proliferate.

It turns out that soluble factors, released in large quantities from certain organs,

can attract circulating cancer cells to take up residence there. Thus, a tumour

needs a certain enviroment for its growth. Besides soluble tissue factors and the

1

2

3 4

5

6

Introduction 4

expression of certain receptors on the tissue-cells, an important role for the

localisation of metastases is played by the pH or the availability of nutrition

factors and other soluble factors. Such signal substances include cytokines,

chemokines, hormones and neurotransmitters. Müller and colleagues have

shown that chemokines stimulate breast cancer cells to carry out the basis

elements of invasion – the cells sent out extensions (pseudopodia), migrated in

a directed manner, and penetrated barriers imposed by the extracellular matrix

[Muller A, et al., 2001]. The receptors for these chemokines are G protein-

coupled receptors, named by their action through guanine-nucleotide-binding

(G) proteins.

Immune system

The immune system protects us from pathogenic exogenic factors and has a

self-control mechanism. We now recognise four broad categories of disease-

causing microorganisms or pathogens: these are viruses, bacteria, fungi, and

other relatively large and complex eucaryotic organisms collectively termed

parasites. The components of the immune system, which protect us against

these pathogens, are the cells that originate from the bone marrow and

maturate in the thymus and in other lymph organs.

To give an overview of the function of the cells of the immune system, we

divided them into the two main categories of white blood cells. First, the myeloid

progenitor is the precursor of the granulocytes, macrophages, dendritic cells,

and mast cells. There are three types of granulocytes which are relatively short

lived and are produced in increased numbers during an immune response,

Introduction 5

when they leave the blood in order to migrate to sites of infection or

inflammation. The neutrophils are phagocytic cells of the immune system and

the most important component of the innate immune response. Eosinophils are

important in the defence against parasitic infection and are increased during

infection. Basophils have a function similar to eosinophils.

Monocytes circulate in the blood and differentiate to macrophages when they

migrate into the tissue. Macrophages are one of three types of phagocytes in

the immune system and play an important role in both the innate and the

adaptive immune response: they release a number of chemokines and

cytokines in order to activate cells of both parts of the immune system.

Moreover, macrophages release substances that have an effect on the vascular

permeability, thus protecting mucosal surfaces against pathogens.

Dendritic cells reside in the tissue, absorb antigens and present them via MHC

(major histocompatibility complex) molecules. This antigen presentation is

essential for the recognition by lymphocytes in the lymph nodes. Dendritic cells

are therefore called professional antigen presenting cells (pAPCs).

The other main category of white blood cells, the lymphoid progenitor, is a

precursor of B lymphocytes, T lymphocytes and natural killer cells.

B lymphocytes differentiate into plasma cells when activated and produce

antibodies for a humoral immune response. T lymphocytes are functionally

divided into T helper cells (CD4+), which activate other cells (B lymphocytes and

macrophages) and cytotoxic T lymphocytes (CD8+), which kill cells infected with

viruses. Futhermore, CD8+ cells are important for the recognition of transformed

cells and thus for the protection against cancer. Natural killer (NK) cells

recognise abnormal cells such as tumours and virus-infected cells, and kill

Introduction 6

them, too. In contrast to cytotoxic T lymphocytes, NK cells recognise the

downregulation of MHC molecules, which is an important mechanism of viruses

and tumour cells to escape from specific immune recognition.

The lymph organs are part of the lymphatic system. The collectivity of this

lymphatic system consists of the lymph vessels, lymph nodes, spleen, thymus

gland, Peyer-Plaques and the pharyngeal tonsils. In the primary lymph organs

like thymus and bone marrow the lymphatic stem cells differentiate into

immunocompetent leukocytes. Spleen, lymph nodes, Peyer-Plaques and

Waldeyer’s ring of the throat (W-Ring) are secondary lymph organs. The

function of these secondary organs is to bring the different types of leukocytes

together, in order to facilitate the direct contact of the cells, which is important

for a coordinated immune response.

The first important step of the adaptive or specific immune response is the

uptake of an antigen by antigen presenting cells (APCs), like macrophages and

dendritic cells, when the pathogens enter the body (Fig. 2). Phagocytic

macrophages deliver an activating or so-called “danger signal” to the dendritic

cells, which makes the cells migrate to the secondary lymphatic organs and

activate T helper cells. B lymphocytes circulating in the blood vessels migrate

into the secondary lymphatic organs, where they interact with the activated

T helper cells, and are in turn activated themselves. When B lymphocytes that

had contact with their specific antigen get a costimmulatory signal from T helper

cells, the B lymphocytes proliferate and differentiate to plasma cells. These

plasma cells produce antibodies, which bind to the pathogen with their two

variable domains and bind to neutrophil granulocytes with the constant region of

Introduction 7

the molecule. Neutrophil granulocytes ingest and destroy the extracellular

pathogen. The defence of intracellular pathogens is predominantly managed by

cytotoxic T cells. Dendritic cells in the lymph nodes, similar to T helper cells,

activate these cells. In addition, the T helper cells provide the cytotoxic

T lymphocytes with interleukin-2, since T helper cells, but not cytotoxic T cells,

produce this T cell proliferation factor. After activation, the cytotoxic

T lymphocytes kill those cells that present the intracellular antigen on their MHC

class I molecules [Parkin J, Cohen B, 2001].

Introduction 8

DC

MΦ

DC

DC

TC

B

DC

IL 2

NG

1.

2. 3.

4.

4.5.6.

7.8.

9.

10.

11.

12.

TH

TH

TH

TC

Figure 2: Migration as part of the immune response. Intracellular pathogens like viruses(hexagons) and extracellular pathogens like bacteria (ovals in grey) enter the body and arephagocytized by dentritic cells and macrophages (1.). The macrophages deliver an activatingdanger signal to dendritic cells (2.), which migrate in the secondary lymphatic organs (dottedarea, 3.). In the secondary lymphatic organs the dendritic cells activate T helper cells (4.).These cells deliver the second signal for activating the B cells (5.), which then produceantibodies (6.). The antibodies bind to pathogens (7.) and work as an opsonising linker forneutrophil granulocytes (8.), which are able to phagocytize the pathogen. IL-2 release of the Thelper cells works as an activator for cytotoxic T cells (9.). These cells recognise (10.) and killinfected cells of the body (11.). At last to complete the circle of the immune response, T helpercells activate macrophages (12.).

This short overview shows that the migration of leukocytes is an essential part

of the immune response. Dendritic cells migrate into lymph nodes after antigen

uptake, neutrophil granulocytes are attracted to sites of bacterial contamination

and inflammation (e.g. by chemokines released by macrophages such as

interleukin-8 or by bacterial metabolic products such as formylated peptides)

and cytotoxic T lymphocytes are attracted to sites of viral contamination by

interferon-inducible chemokines [Entschladen F, et al., 2000].

Introduction 9

Cell migration

The first two chapters of this introduction show that the migration of cells plays

an important role in physiological processes such as an immune response and

pathological processes such as invasion and metastasis development in cancer

disease. Moreover, the short overview (Fig. 3) on the signal transduction of

these processes demonstrates the important role of several diverse signal

substances for the regulation of migrating activity, i.e. the matrix (ECM),

cytokines, and ligands to GPCRs.

Figure 3: Signal transduction pathways of migration. (Modified from F. Entschladen[Entschladen F, Zanker K S, 2000]) The figure shows possible signal transduction pathways oftumour cells, T lymphocytes and neutrophil granulocytes. Migration of these three cell types isinducible by agonists of serpentine receptors. Depending on the signalling pathway, serpentinesignalling leads to PKA-mediated uptake of calcium into the endoplasmatic reticulum or theactivation of phospholipase C with a release of calcium from intracellular stores. For moredetails and explanations see text.

α βγ

α

βγ

α

PKC

CalciumERMARCKS

Gelsolin

PIP2

IP3+DAG

PLCβ1

ATP

cAMP

AC

PKA

FAK

srcPTK

PIP2

IP3+DAG

PLCγ Profilin

PI3K

PIP2

IP3+DAG

PLCβ2

PIP3

SH2

SH3

Vin

PIP2

P-Tyr

ECMChemoattractantsChemokinesCatecholamines

VASP

Profilin

β

Chen & Guan 94Guinebault et al. 95 PaxillinPro

Hynes 92

Clark & Brugge 95Jokusch et al. 95

Jokusch et al. 95Stossel 89

Jokusch et al. 95

Jokusch et al. 95

CD45

Clark & Brugge 95Ledbetter et al. 91 0

C

B

A

GDI GEF

Rho/Cdc42

PKC

Arai & Charo 96Thivierhge et al. 99

Howard et al. 96

Calcium

Berridge & Irvine 94

Aderem 92Blackshear 93

SERCA3Clapham 95

PLB Lin et al. 97

Integrins

RTK

7H(EGF-R) (erbB2)

Cytokines CD100L

Boumsell 848.16Matthes 848.17

β-Arrestin

srcPTK

Lefkowitz et al. 98

Pantaloni et al. 2001WASP

Arp 2/3

Myosin Light Chain

ROCK

α-actinin

PIP2

MLCK

Introduction 10

The initial step for migration – step zero

Every cell has a number of different receptors for the communication with and

recognition of the surrounding area. One of these initial steps of communication

for the initiation of migration is delivered by the extracellular matrix (ECM) via

integrins [Maaser K, et al., 1999]. In collagen matrices, the migratory activity of

T lymphocytes comprises about 30% of a population purified from human

peripheral blood, with great individual varieties between different blood donors.

Neutrophil granulocytes show less interindividual differences. Their matrix-

induced locomotor activity constantly reaches 10% of the population purified

from human peripheral blood. Dendritic cells derived from monocytes by 7-day

cultivation with the granulocyte/macrophage-colony stimulating factor and

interleukin-4 nearly reach 20% locomotor activity [Entschladen F, Zanker K S,

2000].

Tumour cells are very heterogeneously in the matrix-induced locomotor activity.

The locomotor activity reaches from 10% in SW 620, a lymph node metastasis

of a colon-adenocarcinoma, to 36% in SW 480, a primary colon

adenocarcinoma [Kubens B S, Zanker K S, 1998], and to a high locomotor

activity of 80% in MV3, a human melanoma cell line [Friedl P, et al., 1997]. The

migratory potential of a particular tumour cell line has been shown to depend on

the quality and quantity of β1 integrin expression. The pattern of integrin

expression varies between the cells of different tumours and is even different

between cells of the primary tumour and metastases of the same origin [Aplin A

E, et al., 1999].

Introduction 11

Another initial signal is delivered by cytokines and chemokines (chemoattractant

cytokines). Cytokines are a structurally heterogeneous group of small proteins

(<200 AA) known since the 1970s. They have various functions on distinct cells

of the immune system and somatic cells [Kishimoto T, et al., 1994] like

proliferation, differentiation, and apoptosis. Some cytokines, EGF and insulin for

example, bind to a family of class I receptor tyrosine kinases (RTKs) and can

induce migration in tumour cell lines [Dittmar T, et al., 2002].

Over 50 chemokines have been identified today. Chemokines are a large

superfamily of mostly small, secreted chemokines that function in leukocyte

trafficking, recruitment and activation. They play a critical role in many normal

and pathophysiological processes such as allergic responses, infections and

autoimmune deseases, angiogenesis, inflammation, tumour growth and

metastasis, and hematopoiesis. Chemokines are divided into four subfamilies

based on conserved amino acid sequence motifs. Most family members have at

least four conserved cysteine residues that form two intramolecular disulfid

bonds. The subfamilies are defined by the position of the first two cysteine (C)

residues. The CXC (α) subfamily has one AA, the lone CX3C (δ) subfamily

member has three intervening AA, and the CC (β) subfamily has no intervening

amino acids separating the first two cysteine residues. The C (γ) subfamily lacks

the first and third cystein residues [Luster A D, 1998]. Only CXC chemokines

are able to activate neutrophil granulocytes and CC chemokines primarily

stimulate monocytes and lymphocytes. The migration of NK cells is induced by

both CC and CXC chemokines. Lymphotactin (SCM-1α) and SCM-1β are

currently the only two family members of the C chemokine subfamily. Both have

chemotactic activity for lymphocytes and NK cells. Fractalkine, or CX3C, is a

Introduction 12

transmembrane protein with a chemokine domain attached to a long mucine-

like stalk. Membrane bound fractalkine has been shown to promote adhesion of

leukocytes. The soluble chemokine domain of human fractalkine is chemotactic

for T cells and monocytes.

All known chemokine receptors belong to the family known as serpentines or

seven-helices receptors that mediate the biological activities of chemokines.

Most of these receptors exhibit promiscuous binding properties whereby several

chemokines communicate through the same receptor. They are named

according to the chemokine subfamily they bind to [Kelvin D J, et al., 1993].

There are other chemoattactans, not member of any chemokine subfamily, that

bind to serpentine receptors, e.g. formylated peptides such as formyl-methionyl-

leucyl-phenylalanin (fMLP), the complement fragment C5a, or leukotriene B4.

Key regulators of the signal transduction for migration

There are three key regulators for the signal transduction of migration (Fig. 3).

One is the protein kinase A (PKA), which is activated from the second

messenger cyclic adenosinemonophosphate (cAMP) produced from G protein-

coupled transmembrane receptors activated adenylyl cyclase (AC). The γ,β

subtypes of phospholipase C (PLCγ,β) receive their activation signals from

G protein-coupled receptors and integrin receptors in an indirect or direct way

(Fig. 3). PLCs catalyse the breakdown of phosphatidylinositol-4,5-phosphate

(PIP2) into the second messengers diacylglycerol (DAG) and inositol-1,4,5-

phosphate (IP3). DAG in turn activates the protein kinase C (PKC), which is the

second of the three key regulators (Fig. 3). The PKCα is a central regulatory

Introduction 13

molecule in the migration of tumour cells; the migratory activity of a tumour cell

line depends on its level of expression, and the inhibition of this enzyme

completely abolishes migratory activity [Masur K, et al., 2001b]. Most

importantly, the direct link of the PKCα to β1 integrins is crucial for a

chemotactic migration of tumour cells [Parsons M, et al., 2002]. Furthermore,

the PKCα phosphorylates the focal adhesion kinase (FAK), and actin-regulating

proteins such as the myristoylated alanine-rich C kinase substrate (MARCKS)

[Luo B, et al., 2003].

The PKA and the PLC have relevance in the calcium cycling [Lang K, et al.,

2002]; calcium is the third key regulator for migration. The cAMP-dependent

PKA phosporylates phospholamban (PLB), which is an inhibitory regulator for

the calcium pump SERCA (sarcoplasmatic/endoplasmatic reticulum calcium

ATPase), SERCA pumps cytosolic calcium into intracellular stores [Paul R J,

1998]. The PLC mediated IP3 generation in contrast opens these intracellular

stores and thereby causes calcium to be released from the endoplasmatic

reticulum. Calcium as a second messenger is the major substance for the

regulatory signal transduction of migration. An interruption of the above

described calcium cycling [Lang K, et al., 2002] results in the loss of locomotor

activity.

Inducers for migration

As described above at the initial step for migration, we see that a great number

of ligands as cytokines and chemokines are known for the induced migration in

leukocytes and tumour cells. One of these chemokines for migration is the

Introduction 14

stromal cell-derived factor-1 (SDF-1), a CXC chemokine, constitutively

expressed and produced by bone marrow stromal cells; the receptor for SDF-1

is CXCR4. SDF-1 is an inducer for CD4+ lymphocytes [Kijima T, et al., 2002,

Nanki T, Lipsky P E, 2000] and increases the recruitment of non-locomoting

cytotoxic T lymphocytes from 20% to 65% [Entschladen F, et al., 2000]. This

chemokine does not only induce migration, but also delivers a localisation signal

for the development of metastases, as was shown by Muller and co-workers for

the development of breast cancer metastases in the mouse [Muller A, et al.,

2001].

In bacteria, polypeptide synthesis starts with a modified amino acid, i.e. formyl-

methionyl (fMet). fMLP is a synthetic peptide that mimics the activity of

bacterially-derived peptides with its formylated N-terminal methionine groups.

fMLP binds to a serpentine receptor similar to chemokines and is a strong

regulator for the migration of neutrophil granulocytes. It leads to an increase in

the recruitment of non-locomoting neutrophil granulocytes from 14% to 67%

[Entschladen F, et al., 2000].

Neurotransmitters are a further important group of serpentine receptor ligands

with regulating function in cell migration. Norepinephrine, a neurotransmitter

also known as noradrenaline, is a catecholamine neurotransmitter hormone

released from the adrenal glands that has effects on parts of the human brain

where attention and impulsivity are controlled. This substance influences the

fight-or-flight response by activating the sympathetic nervous system to directly

increase heart rate, release energy from fat and increase muscle readiness.

The brain stem called the locus ceruleus is the origin of most norepinephrine

pathways in the brain. Neurons using norepinephrine as their neurotransmitter

Introduction 15

project bilaterally from the locus ceruleus along distinct pathways to the cerebral

cortex, limbic system, and the spinal cord, among other projections.

Norepinephrine is synthesised by a series of enzymatic processes (with the

enzymes tyrosinhydroxylase, dopadecarboxylase and finally dopamine-β-

hydroxylase) in the adrenal medulla that convert tyrosine first to

dihydroxyphenylalanine (DOPA), then to dopamine, which is then

biosynthesised into norepinephrine. Some norepinephrine may then be further

converted to epinephrine.

Norepinephrine is a strong inducer for SW 480 colon carcinoma cell migration

[Masur K, et al., 2001a] and for MDA-MB-468 breast carcinoma cells [Drell IV T

L, et al., 2003]. Furthermore, norepinephrine regulates the migration and

cytotoxicity in NK cells. The locomotion of these cells increases from 16% to

23% [Lang K, et al., 2003].

The aforementioned ligands to serpentine receptors are the strongest inducers

for migration in our hands, respectively, SDF-1 for T lymphocytes, fMLP for

neutrophil granulocytes, and norepinephrine for tumour cells and were thus

used throughout this work to stimulate cell migration.

Neurotransmitters with inhibitory function

GABA

γ-Aminobutyric acid (GABA) is a substance ubiquitously found in bacteria,

yeast, vertebrates and also in plants [Bouche N, et al., 2003]. This non-protein

Introduction 16

amino acid is mainly metabolised through a short pathway. The pathway is

composed of three enzymes: the cytosolic glutamate decarboxilase (GAD), and

the mitochondrial enzymes GABA transaminase (GABA-T) and succinic

semialdehyde dehydrogenase (SSADH). Studies of GABA in vertebrates have

concentrated mainly on its role as a neurotransmitter. GABA activates

ionotropic (ion channels) GABAA and GABAC and metabotropic (serpentine)

GABAB-receptors. The Cl- channels GABAA and GABAC are heteromeric

complexes composed of five subunits, each subunit with four transmembrane

domains. The GABAB-receptor is a serpentine receptor coupled to Ca2+ or K+

channels or adenylate cyclase via Gi/o GTP binding proteins [Behar T N, et al.,

2001]. Functional receptors are formed only after heterodimerisation of

GABA(B1) and GABA(B2) (previously known as GBR1 and GBR2) by interaction

through their C-termini, the first time that this form of 1:1 stochiometry has been

identified within the family of serpentine receptors. Both subunits are members

of the 7-transmembrane receptor family and have over 30% sequence

homology to the metabotropic glutamate receptors. A number of splice variants

have been identified for both GABA(B1) and GABA(B2) [Martin I L, 2002]. The

GABAB receptor is selectively activated by baclofen (4-amino-3-(-4-

chlorphenyl)-butyric acid) [Bormann J, 2000]. This myotonolyticum (Lioresal,

Ciba, Wehr) acts on the central nervous system to relieve spasms, cramping,

and tightness of muscles caused by spasticity in multiple sclerosis or certain

injuries to the spine. There is evidence that GABAB-receptor agonist may be

useful in the treatment of pain and to reduce the craving for drugs in addiction.

The association of malignancy with elevated diamine oxidase (DAO) levels, an

enzyme producing γ-aminobutyric acid (GABA) from putrescine, is well

documented. In ovarian cancer, increased DAO occurs in the malignant tissues

Introduction 17

and plasma. Since higher DAO levels cause GABA accumulation, elevated

GABA concentrations occur in ovarian cancer and are reflected in the urine

[Nicholson-Guthrie C S, et al., 2001].

Anandamide

Cannabinoids are a class of hydrophobic substances found in Cannabis sativa.

The most prominent substrate is Δ-9-tetrahydrocannabinol, which induces

psychoactive effects upon intake. The fervent search for a specific cannabinoid

receptor ended in 1988, when the existence of a specific receptor in the rat

brain was confirmed. Devane and colleagues gave it the designation CB1–R

[Devane W A, et al., 1988]. A second receptor, the peripheral cannabinoid

receptor CB2-R, was discovered in macrophages of the margin zone of the

spleen, and was cloned by Munro and co-workers [Munro S, et al., 1993]. This

CB2-R has since been found in lymph nodes, Peyer-Plaques of the small

intestine, and leukocytes. Both receptors CB1-R and CB2-R are expressed on

leukocytes [Klein T W, et al., 2003, Nong L, et al., 2001, Roth M D, et al., 2002,

Yuan M, et al., 2002].

Both cannabinoid receptors are Gi/o protein-coupled transmembrane receptors,

and the subsequent signaling pathways negatively regulate the adenylyl cyclase

and activate the mitogen-activated protein kinase [Matsuda L A, et al., 1990,

Pertwee R G, 1999]. The discovery of these receptors led to the discovery of

the first endogenous ligand to the CB1-R shortly thereafter, which was found in

pigeon brain [Devane W A, et al., 1992]. This ligand was termed “anandamide,”

based on ”ananda,” the Sanskrit word for bliss, and its amide-containing

Introduction 18

chemical structure. This ligand is an arachidonic acid derivate, also called

arachidonoylethanolamide. Anandamide binds to the CB2-R, as well, but with

less affinity than to the CB1-R [Slipetz D M, et al., 1995]. In accordance with the

aforementioned intracellular signal transduction pathways activated by CB-R

engagement, anandamide inhibits the forskolin-stimulated adenylyl cyclase,

thereby reducing the cellular cAMP production [Slipetz D M, et al., 1995].

In human breast cancer and prostate cancer cells, anandamide inhibits

proliferation via the CB1-R [De Petrocellis L, et al., 1998, Melck D, et al., 2000].

The CB1-R engagement leads to an inhibition of cAMP generation, as described

above, and leads to the suppression of receptor tyrosine kinase signaling, as

was shown by the inhibition of prolactin- and nerve growth factor-induced cell

proliferation [De Petrocellis L, et al., 1998]. With regard to the immune system,

anandamide has an anti-inflammatory function and plays a role in the reduction

of chronic pain. For example, anandamide was found to inhibit neutrophil

recruitment [Berdyshev E, et al., 1998]. Furthermore, there is an inhibitory effect

of anandamide in bronchoalveolar lavage fluid of lipopolysaccharide-treated

mice on tumour necrosis factor α production [Berdyshev E, et al., 1998].

Macrophages themselves produce anandamide and related substances, e.g.

palmitoyl ethanolamide (PEA) and 2-arachidonoyl glycerol (2-AG), upon

stimulation with ionomycin, and also contribute to the homeostasis of

endocannabinoids by inactivating these substances through several pathways

[De Petrocellis L, et al., 2000].

Introduction 19

G protein-coupled receptors

All ligands described above and discussed in the following text bind to

serpentine receptors. Serpentine receptors are molecules with seven

transmembrane helices. They are intracellularly coupled to heterotrimeric

G proteins [Arai H, Charo I F, 1996] and to protein tyrosine kinases (PTK) via β-

arrestin [Luttrell L M, et al., 1999] (Fig. 3, page 9). Structural and functional

classification of the G protein oligomers has been defined by the α-subunits. As

might be expected from proteins that perform certain highly conserved functions

(for example, association with activated hormone receptors, GTP binding and

hydrolysis as well as association with βγ-dimers), the primary sequence of all

known Gα subunits contains 20% invariant conserved amino acids. Outside of

these regions, the sequences of the G proteins are diverse. Four families of

these proteins, termed Gs, Gi, Gq, and G12/13, have been proposed based on

amino acid sequence comparisons. The Gαs class contains Gαs and Gαolf,

which are 88% identical [Jones D T, Reed R R, 1989]. Both proteins activate

the AC and are substrates for ADP-ribosylation catalysed by the A1 protomer of

a toxin synthesised by Vibrio cholera (i.e., cholera toxin). This posttranslational

modification inhibits the intrinsic GTPase activity of the G proteins [Jones D T,

Reed R R, 1989].

The Gαi class contains Gαi-1, Gαi-2, Gαi-3, the retinal Gα, Gαt, two forms of the

brain-specific Gα subunit Gαo-1 and Gαo-2, as well as Gαz. All members of this

class (with the exception of Gαz) contain a conserved COOH-terminal cysteine

residue that is the site of ADP-ribosylation catalyzed by a toxin produced by

Introduction 20

Bortadella pertussis (i.e., pertussis toxin). This irreversible, covalent

modification uncouples the G protein from its activating receptor. Blockade of

cellular responses to stimulation by pertussis toxin treatment has been an

effective experimental procedure employed to implicate this class of Gα

subunits in specific cellular signaling processes. The Gα t subunit activates

retinal cGMP phosphodiesterase, the major effector in vertebrate

phototransduction. Members of the Gαi and Gαo subfamilies are implicated in

the regulation of ion channel activity and regulation of PLC, whereas the

function of Gαz is not known.

The Gαq class contains five family members, Gα11, Gα14, Gα15, Gα16, and Gαq.

These closely related proteins are substrates for neither cholera toxin- nor

pertussis toxin-catalysed ADP-ribosylation. The Gαq subunits are notable

regulators of the β-class of phosphoinositide-specific PLC-β. Gαq and Gα11 are

widely expressed in mammalian tissues. The expression of other members of

the Gαq class, in contrast, is restricted to stromal and epithelial cells as well as

to cells of the hematopoietic lineage. These Gα subunits also activate PLC-β

isoforms and may exhibit a preference for members of the PLC-β2 family that

also display a similarly restricted pattern of expression [Gutowski S, et al.,

1991].

The final class of pertussis toxin- and cholera toxin resistant Gα subunits

contains two proteins, Gα12 and Gα13. The functions of Gα12 and Gα13 have not

been clearly defined. Overexpression of activated forms of these proteins

transforms fibroblasts. Expression and activation of Gα12 and Gα13 occurs in

differentiation of P19 embryonic stem cells in response to retinoic acid.

Activation of Gα13 leads to selective activation of mitogen-activated protein

Introduction 21

kinases, especially jun NH2-terminal kinases [Jho E H, Malbon C C, 1997].

Other data implicate Gα12 and Gα 13 in regulation of the Na+/Cl- antiporter

activity [Morris A J, Malbon C C, 1999].

As I have described above, the Gαs class proteins activate all AC-subtypes (I-

VI), but the enzymes of AC differ in their susceptibilities to regulation by βγ-

subunits, by members of the Gαi class, by Ca2+/ calmodulin, and by the PKC.

βγ-Subunits are effective inhibitors of the type I enzyme, but stimulate activity of

the type II and type IV enzymes in a manner that is highly conditional on

costimulation by Gαs. It is noteworthy that as with other βγ-subunit-dependent

phenomena, stimulation of type II AC requires considerably higher

concentrations of βγ-subunits than activating concentrations of α-subunits.

Thus, abundant Gi heterotrimers are likely to be the physiologically important

source of βγ-subunits for this mode of regulation of AC.

Although the Gi family was initially identified as the G proteins responsible for

inhibition of AC activity, mechanisms proposed for the inhibitory mode of

regulation have been the focus of intense debate. A failure to observe inhibition

of adenylyl cyclase activity by isolated Gαi led to the proposition that

sequestration of Gαs by βγ-subunits might be the mechanism underlying the

inhibitory response. More recent investigations reveal that all three isoforms of

Gαi are equally effective inhibitors of the types V and VI enzymes. Type I AC is

selectively inhibited by Gαo, whereas the types I and V enzymes can be

inhibited by Gαz [Morris A J, Malbon C C, 1999].

Introduction 22

The aims of this study

The migration of tumour cells is a prerequisite for the development of

metastases.

Ninety-five percent of the patients that die from cancer do not die from the

primary tumour, but from the metastases. Evidence is growing that the

migration of tumour cells is not solely a consequence of genetic alterations, but

is regulated by a multitude of epigenetic factors. Chemokines,

neurotransmitters, and other structurally non-related ligands of serpentine

receptors are known as important initiators of migratory activity.

The general aim we followed herein was to distinguish tumour cell migration

from the migration of immune cells in order to interfere selectively with the

tumour cell migration without hindering the immune system. Therefore, we have

to understand the signal transduction pathways and regulatory mechanisms that

initiate and inhibit, maintain, and direct cell locomotion.

Thus, we investigated the initial and inhibitory steps of migation of two different

effector cell types of the immune system, the neutrophil granulocytes and the

T lymphocytes, and compared the migration of these cells to the migratory

behaviour of carcinoma cell lines of the colon and the breast. In particular,

concerning the regulation of migration we investigated known neurotransmitters

on these cells and the subsequent signal transduction pathways that induce and

inhibit migration in the different cell types. Concerning the maintenance of

locomotor activity we investigated the effector signal transduction elements for

the production of motile forces (including calcium and actin).

Experimental procedures 23

Experimental procedures

Material

Table of pharmacological substances used in this work

Substance Distributor

(-)-Arterenol free base (Norepinephrine) Sigma-Aldrich Chemie

GmbH, Taufkirchen,

Germany

(±)-Baclofen Calbiochem, La Jolla, CA

Adenosin 3`,5`-cyclic Monophosphate, N6,O2`-

Dibutyryl-Sodium Salt (db-cAMP)

Calbiochem, La Jolla, CA

Arachidonoylethanolamide (Anandamide) Biotrend Chemikalien

GmbH, Köln, Germany

Cholera Toxin from Vibrio cholerae (CTX) Sigma-Aldrich Chemie

GmbH, Taufkirchen,

Germany

N-(2-Hydroxyethyl)-

7Z, 10Z, 13Z, 16Z-docosatetraenamide (DEA)

Biotrend Chemikalien


Forskolin, Coleus forskohlii Calbiochem, La Jolla, CA

formyl-methionyl-leucyl-phenylalanine (fMLP) Sigma-Aldrich Chemie

GmbH, Taufkirchen,

Germany

Ionomycin, Calcium Salt,

Streptomyces conglobatus

Calbiochem, La Jolla, CA


Substance Distributor

Isoguvacine hydrochloride (Isoguvacine) Sigma-Aldrich Chemie

GmbH, Taufkirchen,

Germany

11-OH-Δ 8-tetrahydrocannabinol-dimethylheptyl

(HU 210)

Biotrend Chemikalien


JWH 133 Biotrend Chemikalien


Muscimol Biotrend Chemikalien


Pertussis toxin from Bordetella pertussis

(PTX)

Sigma-Aldrich Chemie

GmbH, Taufkirchen,

Germany

Stromal cell-derived factor 1 (SDF-1) Biotrend Chemikalien


γ-Aminobutyric acid (GABA) Calbiochem, La Jolla, CA


Equipment

Electronic device Classification Company

Confocal laser

scanning

microscope

Leica TCS 4D Leica Microsystems Vertrieb

GmbH, Bensheim

FACS FACS Calibur BD Becton Dickinson, Heidelberg

Microscope Leica DM IL Leica Microsystems Vertrieb

GmbH, Bensheim

Videocamera Color Video Camera

TK-C1381

JVC DEUTSCHLAND GMBH,

Friedberg

Videorecorder TIMELAPSE SR-

9080EK

JVC DEUTSCHLAND GMBH,

Friedberg

Post processing

computer

Apple Macintosh

Computer

Apple Computer Inc.

Centrifuge Rotina 46 R Hettich, Andreas GmbH & CO.KG,

Tuttlingen

RT-PCR ABI PRISM 7700

Sequence Detector

AB Applied Biosystems, Darmstadt

ELISA-Reader Microplate Reader

Model 550

BIO-RAD Laboratories GmbH,

München


Cell cultivation and isolation

Tumour cell lines

Two cell lines were used in the experiments: The tumour cell line SW 480

originated from a tumour of human colon epithelium (American Type Culture

Collection, Rockville, MD), and was transfected with pEGFP-Actin vector from

Becton Dickinson Clontech, Germany. The SW 480 colon carcinoma cell line

was cultured in L15 (PAA, Linz, Austria) containing 10% active fetal calf serum

and was kept at 37° C without CO2 addition [Masur K, et al., 2001a]. The human

breast carcinoma cell line MDA-MB-468 (American Type Culture Collection,

Rockville, MD) was kept in DMEM culture medium (PAA, Linz, Austria)

containing 10% active fetal calf serum (Boehringer Mannheim Corp., Mannheim

Germany) at 37° C humidified atmosphere with 5% CO2 [Drell IV T L, et al.,

2003].

T lymphocytes

Human CD8+ T lymphocytes were isolated from heparinised peripheral blood.

Using Ficoll-Hypaque (ICN, Meckenheim, Germany) density-gradient

centrifugation, the peripheral blood mononuclear cell fraction was isolated and

the CD8+ T lymphocytes were positively selected using immunomagnetic beads

coated with mouse anti-human CD8 mAbs (Dynabeads, Dynal, Hamburg,


Germany). The cell bound beads were detached with polyclonal anti-mouse Fab

(Detachabead, Dynal, Hamburg, Germany) [Entschladen F, et al., 1997].

Isolated T lymphocytes were maintained overnight in RPMI including 2 mM L-

glutamine, 10% heat-inactivated fetal calf serum (Boehringer Mannheim Corp.,

Mannheim, Germany) and 1% penicillin/streptomycin (50 U/ml and 50 µg/ml;

GIBCO, Eggenstein-Leopoldshafen, Germany)[Entschladen F, et al., 2002].

Neutrophil Granulocytes

The first steps of the neutrophil granulocytes (NG) separation was the same as

described above for the separation of the CD8+ T lymphocytes. After the Ficoll-

Hypaque density-gradient centrifugation the pellet containing erythrocytes and

NG was diluted with platelet-depleted serum, and mixed at 1:1.3 with Matrodex

(Longostil 70, Longostil 40 1:1, Fresenius Kabi, Bad Homburg, Germany)

containing 0.06 M EDTA. After 3 h the NG had settled down and remaining

erythrocytes were remove by a hypotonic lysis with 0.3% sodium chloride for

two minutes on ice. The purified NG were used immediately after isolation

[Entschladen F, et al., 2000].


Cell migration assay

Migration chambers were constructed using a microscope slide; wax walls were

applied on three sides and a cover slip was mounted on top (Fig. 4) [Drell IV T

L, et al., 2003].

Figure 4: Schema of a migration chamber. A normal microscope-slide in size of 76 x 26 mm2

was used as the basis of the chamber.

The resulting chamber was filled with a mixture of 50 µl cell suspension with

100 µl collagen solution. The collagen solution was made of 5.5 µl bicarbonate,

11.1 µl minimum essential Eagle’s medium (Flow, McLean, VA) and 83.4 µl

Vitrogen (Cohesion, Palo Alto, CA) containing 95-98% collagen type I with the

remainder being comprised of type III collagen. The applied amount of cells was

dependent on the cell type. The concentration of tumour cells was 4 x 105

cells/ml, the number of immune competent cells was 3 x 106/ml. After filling the

chambers the gel were allowed to polymerise for 30 min at 37° C. In order to

investigate the regulation of cell migration, the substances (pharmaceuticals


and ligands to serpentine receptors, i.e. SDF-1, fMLP or norepinephrine,

respectively) were mixed with the collagen solution prior to polymerisation, and

the residual chamber volume was filled with solutions of these substances after

polymerisation.

The locomotor behaviour of cells in the chambers was recorded via time-lapse

videomicroscopy for more than 12 h for tumour cells or for 1 h for cells of the

immune system. The experimental setup of the time-lapse videomicroscopy is

shown below (Fig.5).

Figure 5: Time-lapse video microscopy: Heating unit, microscope with camera and time-lapse videorecorder.

The migration chamber was placed under a box, which was heated

continuously with special lamps on a temperature of 37° C. The movement of

the cells was recorded with a videocamera connected to a microscope (Leica

Microsystems Vertrieb GmbH, Bensheim, Germany). The time-lapse recording

was managed via a videorecorder. The amplification was 1:200. The recorded

time-lapse was 1920 for carcinoma cell lines and 80 for immunocompetent

cells.

time-lapse

Video-Camera

37°C37°C


The post processing was managed with a computer. The so-called cell tracking

was a digitalisation of the paths of 30 randomly selected cells in steps of 15 min

in the case of tumour cells and 1 min-steps for immune cells. From the x/y data

in combination with the time we calculated the migration activity, displacement,

velocity, speed, directionality, frequency of pauses and duration of pauses.


EGFP-actin vector construction

EGFP (enhanced green fluorescent protein) is a red shifted green fluorescent

variant of GFP. The pEGFP-actin vector encodes a fusion protein consisting of

the fluorescent protein and the gene encoding human cytoplasmic β-actin. The

excitation maximum of the protein is 488 nm and the emission maximum is

507 nm. More information about the sequence of the vector, the restriction map,

and neomycin/kanamycin resistance are shown in Figure 6, or are available on

the information sheet PR08331, published 30 August 2000 by CLONETECH

Laboratories, Inc., Palo Alto USA.

Figure 6: Restriction Map of pEGFP-Actin. The grey vector show the coding sequence ofEGFP followed by the gene encoding human cytoplasmic β-actin. For more details see textabove.


Transformation protocol

We used the Transform AidTM protocol (MBI FERMENTAS GMBH, 68789

St.Leon-Rot, Germany). In short, we incubated bacteria (E.coli) with transfection

medium T and with 1 µl of the DNA (0.1 µg/µl) for 30 min on ice. After a short

heatshock (1.5 min at 42° C) the bacteria were set on ice again for 2 minutes.

After the transfection an inoculate of the bacteria suspension was transferred on

agar-plates. Agar-plates consisted of 16 g/l peptone, 10 g/l yeastextract, 10 g/l

natriumchloride and 15 g/l agar-agar. After overnight incubation at 37° C a cell

clone was picked and transferred in a tube with medium.

Isolation of plasmid DNA

The isolation was performed using the NucleoSpin, plasmid-protocol

(MACHEREY-NAGEL, 52313 Düren, Germany). The bacteria cells were lysed

and centrifuged with a NucleoSpin plasmid column. The DNA was separated

after immobilisation from the column.

The concentration of the nucleic acids was determined by measuring the

extinction of the sample at 260 nm and 280 nm. The extinction of the purine

bases is at 260 nm, contaminating polysaccharides and proteins were

measured with the extinction of 280 nm.


The concentration of the probes was calculated with the formula:

€

E •OD • f1000

= c

We measured an extinction of 0.669 at 260 nm and 0.389 at 280 nm. With a

dilution factor of 100 this results in a concentration of 3.35 µg/µl. The quotient of

the extinction 260/280 nm should be between 1.7 and 2.0. Our result of the

quotient was 1.72.

E = ExtinctionOD = optical density(50 µg/ml for double stranded DNA)f = dilution factorc = concentration of DNA (µg/µl)


The control of the DNA preparation with restriction enzymes

For the control of the DNA purity and correct ligation, 2 µl DNA, 1 µl of each

restriction enzyme BamH I and Xho I, 4 µl buffer (double concentrated) and

12 µl distilled-water were mixed. After 5 min incubation at 37° C the DNA was

applied to agarose electrophoresis (1% agarose with TAE). Two markers were

added with a range from 50 bp up to 10,000 bp (Gene Ruler, 100 bp DNA,

50 bp up to 1,031 bp; Massive Ruler, high range, 1,500 bp up to 10,000 bp).

The electrophoresis was performed in a horizontal chamber with 50-70 V for

30 min with a running buffer of TBE (Tris HCl 100 mM, 83 mM boracid, 1 mM

EDTA, pH 7.5). After electrophoresis the agarose gel was stained with a

solution of ethidiumbromid (1 µg/ml) (Fig. 7).

Figure 7: Picture of a DNA agarose blot. Lane A shows marker “Massiv Ruler high range”from 1,500 to 10,000 bp and lane B is marker “Gene Ruler 100 bp DNA" from 50 to 1,031 bp.The third lane C shows the two restriction fragments of the DNA.


The pEGFP-actin vector is a circular vector of 5,820 bases. The restriction

enzymes BamH I und Xho I cut out a part of 1,136 bases, with the remainder of

4,684 bases. The picture of the agarose gel shows two fragments. One

fragment is between the makerlane 4,000 bp and 5,000 bp and the other DNA

fragment is between 1,031 bp and 1,500 bp confirming the correct expression

and isolation of the plasmid.

Transfection of SW 480 colon carcinoma cells

Transfection of the SW 480 cells was performed based on the protocol of

OligofectAMINETM –reagent from Invitrogen Corporation (Carlsbad, California

92008, USA). According to the protocol of the distributor we plated the cells in a

96 well plate. After 50% confluency of the cells was reached, an oligofect-

complexed plasmid DNA was added to the adherent cells and incubated for

12 h. The cells were cultured with L15 (400 µg/ml G418, 10% FCS) selection

medium. After a week of cultivation we sorted the cells with the flow-cytometer

and obtained a purity of transfected cells of 97% (see Fig. 8).


Figure 8: FACS-analysis of the transfection rate of the SW 480 colon cells: Picture Ashows normal SW 480 colon carcinoma cells. In picture B is the FACS-analysis of thetransfected cells. The bar M1 is set for calculating the transfection rate.

Confocal laser scanning microscopy

The images of the cells were made with the EGFP-actin transfected SW 480

colon adenocarcinoma cells. The transfected cells we incorporated in a 3D-

migration chamber as described in the cell migration assay above. After

polymerisation and closing the chamber with waxcomposide, the slides were

placed under a 37° C heated box and pictures of the green fluorescence light at

507 nm and the transmission light were made in 90 seconds intervals. These

pictures were transfered to a computer and films were created with the program

I-View.

A B


Flow-cytometrical measurement of cytosolic calcium

For the investigation of changes in cytosolic calcium by treatment with

norepinephrine or GABA, 2.5 x 106 SW 480 colon carcinoma cells were loaded

with 2 µM fluo-3/AM (Molecular Probes Europe BV, Leiden, Netherlands). After

30 minutes incubation, the calcium-induced fluo-3/AM fluorescence was

measured immediately after addition of norepinephrine and GABA alone or in

combination, using a FACS Calibur Becton Dickinson (Becton Dickinson GmbH,

Heidelberg, Germany) flow cytometer.

Flow-cytometrical detection of cannabinoid-receptors

The presence of CB1-R and CB2-R has been proven on the surface of

leukocytes [Klein T W, et al., 2003, Nong L, et al., 2001, Roth M D, et al., 2002],

but not on colon carcinoma cells. Therefore, we analyzed the expression of

these two receptors flow-cytometrically using a FACS Calibur flow-cytometer as

above. We incubated 1 x 105 cells with 5 µg/ml of the primary antibody (H-150

for the CB1-R, Santa Cruz Biotechnologies, Santa Cruz, CA, USA; the anti-CB2-

R antibody was derived for Calbiochem) for 10 min at room temperature. After

washing we incubated the cells with 5 µg/ml of a fluorescein-isothiocyanate

(FITC)-conjugated anti-rabbit antibody (Coulter-Immunotech, Hamburg,

Germany). Nonspecific binding was determined by an isotypic control rabbit

antibody (Coulter-Immunotech). In addition, flow-cytometry was used to assess


the viability of the cells immediately after the end of the migration experiments

using propidium iodide staining as described previously [Masur K, et al., 2001a].

Measurement of cellular cAMP

For the measurement of changes of the cellular cAMP concentration, 6 x 104

cells were incubated for 20 minutes at 37° C with either medium alone or with

10 µM norepinephrine, 100 µM GABA, or the combination of both

neurotransmitters. For positive control, the cells were incubated with 500 ng/ml

cholera toxin or 500 ng/ml pertussis toxin (both Sigma, Deisenhofen, Germany)

under the same conditions. After incubation, cells were lysed and the cAMP

level was measured using a cAMP enzyme-linked immunoassay system

(Amersham Pharmacia Biotech, Buckinghamshire, UK) as described by the

manufacturer.

Immunoblotting of protein tyrosine phosphorylation

Changes of the PTK-mediated phosphorylation pattern in tumour cells was

analysed by immunoblotting with anti-phosphotyrosine antibodies. SW 480

colon carcinoma cells (5 x 105) were lysed 10 min at 95° C in Laemmli sample

buffer [Laemmli U K, 1970] after incubation with GABA 100 µM, norepinephrine

10 µM and both together. Using polyacrylamide gel electrophoresis the proteins

were separated according to the method of Laemmli and were transferred to an

Immobilion-P membrane (Millipore, Bedford, MA). The blocking of the

membrane with 5% milk powder over night was followed by a one hour


incubation of the primary phosphotyrosine antibody (1:1000 dilution; Upstate

Biotech, Lake Placid, NY). The membrane was washed vigorously with PBS-

Tween (0,5%) and was incubated with a peroxidase-linked anti-mouse antibody

(1:1000 dilution, Amersham Pharmacia Biotech, Buckinghamshire, UK) for 2 h

at room temperature. A chemiluminescence substrate (Boehringer Mannheim.

Mannheim, Germany) was added for 2 min at room temperature and this signal

was detected by exposure to a Kodak X-OMAT AR film sheet (Sigma-Aldrich,

Deishofen, Germany).

Results 40

Results

All investigated cells, leukocytes as well as tumour cells, developed

spontaneous locomotor activity immediately after incorporation of the cells

within a three-dimensional collagen matrix.

Cell migration regulated by GABA

Effect of GABA on the migration of tumour cells

Norepinephrine induces migration of SW 480 colon carcinoma cells [Masur K, et

al., 2001a] and MDA-MB-468 breast cancer cells [Drell IV T L, et al., 2003]. This

induced tumour cell migration was completely inhibited by GABA (Fig. 9).

Norepinephrine treatment increased migration from 42 ± 13% spontaneously

locomoting cells to 63 ± 3%, whereas GABA alone had no effect (46 ± 4%

locomoting cells), but abolished the norepinephrine-induced migration (37 ± 3%)

in colon carcinoma cells (Fig. 9A). Similarly norepinephrine-induced migration

from 26 ± 11% spontaneously locomoting cells to 44 ± 7% in breast cancer cells

(Fig. 9B), whereas GABA alone had no effect (15 ± 6% locomoting cells), but

abolished the norepinephrine-induced migration (20 ± 2%) in these cells

significantly (p < 0.005).

Results 41

Figure 9: The effect of norepinephrine and GABA on the migration of SW 480 coloncarcinoma cells and MDA-MB-468 breast cancer cells. The migration of the cells wasinduced by addition of 10 µM norepinephrine (NOR; CON = control), and this induced migrationwas blocked with 100 µM of GABA added to SW 480 colon (A) and MDA-MB-468 breast (B)carcinoma cells. The graphs show mean values of three independent experiments (90 cellswere analysed).

As explained in the introduction section, there are two kinds of GABA receptors.

Utilising specific agonists to these receptors, we discovered which of these

receptors acts as an inhibitor of the norepinephrine-induced migration. Using

the specific GABAB-receptor agonist baclofen, we have shown that GABA acts

via these serpentine receptors (Fig. 10) in both tumour cell lines.

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Results 42

Figure 10: The effect of norepinephrine and baclofen on the migration of SW 480 coloncarcinoma cells and MDA-MB-468 breast cancer cells. The migration of the cells wasinduced by addition of 10 µM norepinephrine (NOR; CON = control), and this induced migrationwas blocked with 100 µM of baclofen (BAC) added to SW 480 colon (A) and MDA-MB-468breast (B) carcinoma cells. The graphs show mean values of three independent experiments(90 cells were analysed).

The specific GABAA,C-receptor agonist isoguvacine was used to investigate the

engagement of these receptors in the GABA-mediated inhibition of the

norepinephrine-induced migration of both tumour cell lines. Isoguvacine had no

effect in both cell lines on the spontaneous and induced migration (Fig. 11).

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Results 43

Figure 11: The effect of norepinephrine and isoguvacine on the migration of SW 480colon carcinoma cells and MDA-MB-468 breast cancer cells. The migration of the cells wasinduced by addition of 10 µM norepinephrine (NOR). Addition of 100 µM of isoguvacine (ISO) toSW 480 colon (A) and MDA-MB-468 breast (B) carcinoma cells had no effect on thenorepinephrine-induced migration and spontaneous migration (CON) of these cells. The graphsshow mean values of three independent experiments (90 cells were analysed).

Therefore, the inhibitory effect of GABA in tumour cells is solely mediated via

GABAB-receptors. In search for the molecular mechanisms underlying the

regulation of migratory activity of norepinephrine and GABA, we investigated

the changes of cellular cAMP resulting from AC engagement by

Gsα/Giα proteins. Dibutyryl-cAMP (db-cAMP), a stable non-hydrolysable cAMP-

analogue, reduced the spontaneous migratory activity of SW 480 tumour cells

(27 ± 4% locomoting cells; Fig. 12A). Cells treated with dibutyryl-cAMP in

combination with GABA and norepinephrine developed a migratory activity of

55 ± 18% locomoting cells, which was similar to norepinephrine alone

(55 ± 15% locomoting cells), whereas cells treated with norepinephrine and

GABA revealed a migratory activity of 36 ± 12% locomoting cells (Fig. 12A). In

addition, we directly measured cellular cAMP using an enzyme-linked immuno-

assay (ELISA). Norepinephrine induced an increase of cellular cAMP by 45.2%,

while GABA reduced the cellular cAMP concentration by 48.5% (Fig. 12B). The

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Results 44

addition of GABA to norepinephrine-treated cells significantly reduced the

norepinephrine-induced increase of cAMP. Therefore, an increase of locomotor

activity is coupled to an increase of cellular cAMP, while a reduction of cellular

cAMP decreases migratory activity.

Figure 12: The effect of norepinephrine, dibutyryl-cAMP and GABA on the migration ofSW 480 colon carcinoma cells. (A) The migration of the cells was induced by addition of10 µM norepinephrine (NOR). 100 µM GABA was added to the norepinephrine-induced cellswith and without 1 µM dibutyryl-cAMP (db-cAMP). Db-cAMP (1 µM) alone was added to thecells as a control. The graphs show mean values of three independent experiments (90 cellswere analysed).(B) The role of intracellular cAMP in the regulation of SW 480 colon carcinoma cell migrationwas also analysed by an enzyme-linked immunoassay after treatment with 10 µMnorepinephrine or 100 µM GABA alone or in combination. Statistical significance of the cAMPreduction by GABA in norepinephrine-treated cells was calculated using Student’s T test(p<0.02, indicated by an asterisk). Cholera (CTX) and pertussis (PTX) toxins were used aspositive controls. The diagram shows values ± SD of three independent experiments.

Besides the regulation of the AC via the α subunit of G proteins, the βγ subunit

activates a second pathway via G protein receptor-coupled kinases, β-arrestin

and PTKs of the src family [Luttrell L M, et al., 1999]. Activation of the PTKs

leads to a phosphorylation and activation of the PLCγ, which produces DAG

from the breakdown of PIP2. DAG in turn is an activator for the PKCα, an

important element in the onset of tumour cell migration [Masur K, et al., 2001b].

We investigated the activity of PTKs after treatment of the tumour cells with

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Results 45

norepinephrine and GABA by analysing the phosphotyrosine pattern of proteins

in whole cell lysates (Fig. 13).

Figure 13: PTK phosphorylation pattern of SW 480colon carcinoma cells after incubation with GABAand norepinephrine and in combination. Lane A:control cells without neurotransmitter treatment. LaneB shows the phoshorylation pattern after incubationwith GABA and lane C shows the phosphorylationpattern with norepinephrine incubation. In lane Dnorepinephrine and GABA were added together. Perlane 5 x 105 cells were applied.

In this blot after one-minute incubation of SW 480 colon carcinoma cells with

GABA 100 µM (B) and norepinephrine 10 µM (C) alone and in combination (D)

no differences in the tyrosine phosphorylation patterns of proteins can be

observed.

As described in the introduction, the PKA is activated by cAMP. One substrate

of the PKA is PLB, which is in turn an inhibitory regulator for the calcium pump

SERCA. This pump sequestrates cytosolic calcium into intracellular stores.

Flow-cytometrical measurement of cytosolic calcium did not show any changes

of the cytosolic calcium concentration after treatment of the SW 480 cells with

GABA, whereas norepinephrine-treatment led to a strong increase of the

cytosolic calcium concentration (Fig. 14). Addition of GABA to norepinephrine-

Results 46

500

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NOR

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* *

treated cells did not change cytosolic calcium as compared to norepinephrine

alone (Fig. 14).

Figure 14: Flow-cytometrical measurementof changes of the intracellular calciumconcentration after treatment withnorepinephrine and GABA. Cells wereloaded with the calcium-dye fluo-3/AM andsubjected to flow-cytometry immediately afteraddition of the neurotransmitters. The diagramshows mean values ± SD of threeindependent experiments. The meanfluorescence intensity of the control wasadjusted to a value approximately 700.Treatment of the cells with 5 µg/ml ionomycinserved as a positive control and led to anincrease to a mean fluorescence intensity of1971 ± 302 (not shown). Statisticallysignificant changes vs. control are indicatedby an asterisk (Student’s T test; p<0.05).

Calcium is crucially involved in the regulation of tumour cell migration by

regulating proteins that interact with the actin network by polymerisation and

depolymerisation [Carlier M F, et al., 1997, Jockusch B M, et al., 1995]. This

actin network was shown in living SW 480 carcinoma cells, transfected with

EGFP-actin, on a confocal microscope (Fig. 15). To see changes in actin

location and concentration, we took pictures every 90 seconds.

In Figure 15 we see a cell with a bipolar cell shape. The orange coloured area

depicts EGFP-actin distribution. In these 6 frames a brighter dot was seen

shifting 10 µm from the left to the right. This dot ended in a newly created

pseudopod arrow.

Results 47

Figure 15: Distribution of actin in migratingSW 480 colon carcinoma cells. Cells weretransfected with EGFP-actin and wereincorporated within 3D collagen lattices. Thefluorescence image was taken using confocalmicroscopy. The white bar in the first picture(upper right) represents a distance of 10 µm.The arrow shows areas of high actinconcentration.

Results 48

Effect of GABA on the migration of leukocytes

In order to elucidate the influence of GABA on the immune response, we

stimulated CD8+ T lymphocytes with 1 µg/ml SDF-1, which is one of the major

stimulatory chemokines for migration. Addition of 1 µM GABA to SDF-1

stimulated cells reduced the SDF-1 effect from 66 ± 16% to 18 ± 24%; GABA

alone had no effect on the spontaneous migration of T lymphocytes (Fig. 16A).

Moreover, GABA had no effect on the migration of 10 nM fMLP stimulated NG

(Fig. 16B).

Figure 16: Effect of GABA (1 µM) on the spontaneous and induced migration of cytotoxicT lymphocytes (A) and neutrophil granulocytes (B). We induced the migratory activity ofT lymphocytes and NG, by using 1 µg/ml SDF-1 and 10 nM fMLP, respectively. The migrationexperiments were performed as described in the tumour cell section, with the exception that2 x 105 cells per sample were used for the leukocyte migration experiments. The figures showmean values of three independent experiments (90 cells were analysed).

As written above, downstream in the signal transduction, G protein-coupled

receptors regulate the AC, which produces the second messenger cAMP. What

influence has direct addition of db-cAMP on the GABA effect? GABA reduces

the SDF-1 stimulated migration of CD8+ T lymphocytes from 82 ± 2% to

65 ± 3%. This reduction in migration was reversed by the addition of 10 µM db-

cAMP to 87 ± 3% (Fig. 17A).

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Results 49

Figure 17: Effect of 1 µM db-cAMP (A) and 10 µM forskolin (B) on the AC in sameconditions as in the previously described experiment (see Fig. 16). We induced themigratory activity of T lymphocytes by using 1 µg/ml SDF-1. FORS mattered 10 µM forskolin.The migration experiments were performed as described previously. The figure shows meanvalues of three independent experiments (90 cells were analysed).

Furthermore, forskolin, a stimulating diterpen for all subtypes of the AC, had no

stimulatory effect on the migration when applied alone (20 ± 3%), or in

combination with 10 µM GABA (23 ± 2% locomoting cells) and 10 µM SDF-1

(Fig. 17B).

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Results 50

Cell migration regulated by anandamide

Effect of anandamide on the migration of tumour cells

Cannabinoid receptors (CB-Rs) are Gi protein-coupled receptors which inhibit

the AC in the same way as GABA receptors. With regard to this correlation we

investigated the engagement of the different CB-receptors with specific receptor

agonists on the norepinephrine-induced migration of SW 480 colon carcinoma

cells.

Figure 18: Ef fects ofanandamide on the migration ofSW 480 colon carcinoma cells.In A to C, the migration of the cellswas induced with 10 µMnorepinephrine. Anandamide (A)or the CB1-R specific agonist DEA(B), were added to the collagenmixture at concentrations of40 nM. The CB2-R specific agonistJWH 133 (C) was used at 10 nM.

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Results 51

We observed that the norepinephrine-induced tumour cell migration was

completely inhibited by anandamide in SW 480 cells (Fig. 18A). Norepinephrine

(10 µM) induced migration from 28 ± 5% spontaneously locomoting cells to

54 ± 8% locomoting cells. Anandamide (40 nM) alone had no effect (29 ± 4%

locomoting cells), but inhibited the norepinephrine-induced migration (36 ± 5%;

p<0.05). We investigated the receptor responsible for this inhibitory function of

anandamide by receptor-specific agonists. We used docosatetraenamide (DEA;

40 nM) as a specific agonist for the CB1-R, and JWH 133 (10 nM) as a specific

agonist for the CB2-R. The differences in the concentrations that were used

result from the different affinities described for the agonists [Barg J, et al., 1995,

Huffman J W, et al., 1999]. DEA (Fig. 18B), but not JWH 133 (Fig. 18C)

inhibited the norepinephrine-induced migration. After treatment with DEA, the

norepinephrine-induced migration was reduced significantly (p<0.005) from

62 ± 4% to 37 ± 5% locomoting cells (Fig. 18B), whereas treatment with

JWH 133 had no effect (60 ± 10% locomoting cells with norepinephrine vs.

62 ± 6% locomoting cells with norepinephrine and JWH 133; Fig. 18C).

Results 52

DEA is a specific agonist for the CB1-R. The presence of CB1-R and CB2-R has

been proven on the surface of leukocytes [Klein T W, et al., 2003, Nong L, et

al., 2001, Roth M D, et al., 2002, Yuan M, et al., 2002], but not on colon

carcinoma cells. Therefore we analysed flow cytometrically the expression of

the two receptors with the anti-CB-R specific antibodies. Non-specific binding

was determined by an isotypic control rabbit antibody.

Figure 19: Expression of cannabinoid receptors on SW 480 colon carcinoma cells. TheFITC-fluorescence of specifically bound antibodies against CB1-R and CB2-R (red area) wascompared to an isotypic control antibody (black line). The mean fluorescence intensity (MFI)was 5.13 in the isotypic control and 7.05 for the CB1-R (A) and for the CB2-R (B) 5.49.

Both CB1-R and CB2-R were expressed on the surface of these cells. The mean

fluorescence intensity (MFI) for the CB1-R to the isotypic control was 7.05 (Fig.

19A), whereas the expression of the CB2-R was very low at 5.49 (Fig. 19B), as

compared to isotype control (5.13).

A B

Results 53

Effect of anandamide on the migration of leukocytes

Furthermore, we observed that the SDF-1-induced migration of T lymphocytes

was inhibited by anandamide, similar to the effect observed in tumour cells (Fig.

20A): the SDF-1-induced migration was significantly reduced from 77 ± 19% to

55 ± 25% (p<0.05) locomoting cells in combination with anandamide, whereas

this endogenous cannabinoid alone had no effect on the migration of the

T lymphocytes. There was no significant difference between the spontaneous

migration (5 ± 3%) of the cells and the cells treated with anandamide (3 ± 1%).

In contrast to our results with SW 480 colon carcinoma cells, the specific CB1-R

agonist DEA did not reduce the SDF-1-induced migratory activity of the

T lymphocytes (Fig. 20B). JWH 133, the specific CB2-R agonist, did however

reduce the SDF-1-induced migration by 20% (from 58 ± 17% to 36 ± 11%;

Fig. 20C).

Results 54

Figure 20: In f luence ofanandamide and specific CB-Ragonists on the SDF-1 inducedmigration of CD8+ T lymphocytes.In each experiment (A-C) we used1 µg/ml SDF-1 to induce migration.Anandamide (ANA; A) and DEA (B)were added at concentrations of40 nM. JWH 133 (JWH; C) wasadded to the collagen at aconcentration of 10 nM. CON is thespontaneous migration (control)without any pharmacologicalsubstance.

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Results 55

Neutrophil granulocytes are an integral part of the innate immune system, and

can recognise the formylated peptide fMLP via a serpentine receptor (fMLP-R).

Neutrophil granulocytes respond to fMLP with an increase of migratory activity

from 6 ± 2% up to 71 ± 4% locomoting cells (Fig. 21) [Entschladen F, et al.,

2000]. The addition of anandamide to these cells had no effect on the

spontaneous locomotion (8 ± 7% locomoting cells), nor on the fMLP induced

migration (68 ± 11% locomoting cells).

Figure 21: Influence of anandamide on the fMLP induced migration of NG. In thisexperiment we used 10 nM fMLP to induce the migration. Anandamide (ANA) was added atconcentrations of 40 nM to the collagen. CON is the control experiment without the addition ofany substance to the collagen.

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Discussion 56

Discussion

The migration of cells is a process of communication with the environment, and

accordingly a lot of inducers for the migration of leukocytes and tumour cells are

known [Azenshtein E, et al., 2002, Dittmar T, et al., 2002, Entschladen F, et al.,

2002, Lang K, et al., 2002, Marino F, et al., 2001, Nanki T, Lipsky P E, 2000].

Two important groups of these inducers are the chemokines and

neurotransmitters, such as SDF-1, fMLP and norepinephrine, which couple to

serpentine receptors and stimulate the migration of several cell types of the

immune system and tumour cells [Bristow M R, et al., 1985, Kijima T, et al.,

2002, Tsu R C, et al., 1997]. In light of the high number of articles describing

substances that induce migration, it is surprising that to date no chemokine or

neurotransmitter has been characterised, which inhibits the migration of these

cells. We have presented herein data on two neurotransmitters with such an

inhibitory function on migration, i.e. GABA and anandamide.

Anandamide alone had no effect on the migration of both tumour cells and

leukocytes. These results are in accordance with findings of Kishimoto and co-

workers on HL-60 leukaemia cells differentiated into macrophage-like cells

using Transwell™ inserts [Kishimoto S, et al., 2003]. In contrast, anandamide

was shown to have a stimulatory effect on embryonic kidney cells transfected

with human CB1-R gene [Song Z H, Zhong M, 2000]. Anandamide in high

concentrations stimulates microglial cell migration [Walter L, et al., 2003], and

migration of myeloid leukaemia cells transfected with the CB2-R gene [Jorda M

A, et al., 2003]. Similar to anandamide, functional differences between neuronal

Discussion 57

cells and leukocytes have been observed by Behar et al. for GABA, too [Behar

T N, et al., 2001]. In our experiments GABA has no stimulatory effect on tumour

cells and leukocytes. However, in our experiments GABA and anandamide

abolish the norepinephrine-induced migration of SW 480 colon and MDA-MB-

468 breast carcinoma cells. These two neurotransmitters bind to several

receptor subtypes, which could inhibit the migration of tumour cells. GABA

binds on three receptor subtypes GABAA, GABAB and GABAC [Bormann J,

2000], anandamide to CB1-and CB2-receptor subtypes [Reggio P H, Traore H,

2000]. With specific agonist to these receptors we were able to analyse the

relevant receptor for the inhibitory signal of migration. The inhibitory function of

GABA on the migration of these tumour cells is mediated by the metabotropic

GABAB-R. Anandamide, or the endogenous specific CB1-R agonist DEA, exert

the same inhibitory effect via the CB1-R. Both receptors are serpentine

receptors coupled to Gi proteins. Gi protein-coupled receptors decrease the

cAMP production by inhibiting the AC [Gilman A G, 1984]. It is well known from

heart muscle cells that binding of norepinephrine leads to activation of

stimulating G (Gs) proteins [Bristow M R, et al., 1985]. These GS proteins

increase the activity of the adenylyl cyclase, which generates the second

messenger cAMP [Gilman A G, 1984]. In our experiments on the regulatory

signal transduction of the norepinephrine- and GABA-mediated effects on cell

migration we focussed on these G protein-mediated regulation of cellular cAMP.

The cellular cAMP, which we measured after treatment of the cells with

norepinephrine and GABA alone prove this stimulating Gs and inhibitory Gi

effect. The cellular cAMP concentration is decreased with GABA and increased

with norepinephrine. The addition of GABA to norepinephrine-treated cells

significantly reduced the norepinephrine-induced increase of cAMP. In our

Discussion 58

migration assay an increase of locomotor activity is coupled to an increase of

cellular cAMP, while a reduction of cellular cAMP decreases migratory activity.

The decrease of migratory activity of GABA- and norepinephrine-treated cells is

abolished by simultaneous db-cAMP addition. Interestingly, tumour cells treated

with db-cAMP alone did not increase migratory activity. This shows that a

decrease of cAMP is a sufficient stop signal for the norepinephrine-induced

migration, but an increase of cAMP is not a sufficient start signal for the

migration of the tumour cells. The aforementioned stimulatory effects of

anandamide, as observed by Jorda and by Song [Jorda M A, et al., 2003, Song

Z H, Zhong M, 2000], might be due to the overexpression of the receptors in the

transfected cells. Such overexpression might lead to the interaction of the

receptor not only to Gi, but also to Gs proteins as was shown on the example of

the α2-adrenoceptor [Eason M G, et al., 1992].

In turn, cAMP binds to multiple effector molecules, which are involved in the

regulation of the cytosolic calcium concentration. A key effector molecule is the

PKA, which phosphorylates PLB. Phosphorylation of PLB leads to its release

from SERCA, which sequestrates cytosolic calcium into intracellular stores

[Paul R J, 1998].

In addition to the G protein-mediated signalling, Luttrell et al. [Luttrell L M, et al.,

1999] described an activation of PTKs via β-arrestin after β2-adrenoceptor-

engagement. We have shown that a PTK-dependent activation of the PLCγ is a

crucial regulator for the norepinephrine-induced migration of colon carcinoma

cells [Masur K, et al., 2001a]. Two second-messengers are generated by the

PLCγ. First IP3, which opens intracellular calcium channels, and second is

diacylglycerol, which activates the PKCα. In this context it is important to stress

Discussion 59

that an activation of the PKC with the diacylglycerol analogue phorbol-12-

myristate-13-acetate (PMA) is alone a sufficient start signal for the induction of

very high locomotor activity in colon carcinoma cells [Masur K, et al., 2001b].

Therefore, the molecular switch to turn migration on can be different from the

molecular switch to turn migration off. In addition, no changes of the protein

tyrosine phosphorylation pattern were observed in cells treated with

norepinephrine and GABA compared to cells treated with norepinephrine alone,

as analysed by anti-phosphotyrosine immunoblotting. This supports the view

that the inhibitory GABA effect is predominantly or solely mediated by the

regulation of cellular cAMP and does not interfere into PTK-mediated PLCγ

activity.

At least, both pathways – the cAMP-regulated adenylyl cyclase activity and the

PTK-induced PLCγ activity lead to the activation or enzymatic generation of

molecules, which regulate the intracellular calcium concentration [Lang K, et al.,

2002]. In T24 breast cancer cells signalling of calcium in migrated cells was

observed with a calcium-dye for living cells. In comparison to non-migrating T24

cells the calcium oscillation is highly increased [Lang K, et al., 2002].

Treatment of the SW 480 cells with GABA did not lead to changes of the

cytosolic calcium concentration, whereas norepinephrine-treatment led to a

strong increase of the cytosolic calcium concentration. The norepinephrine-

effect is caused by the previously described PLCγ activation catalysing the

production of IP3 [Masur K, et al., 2001a], which leads to the fast release of

calcium from the endoplasmatic reticulum (Fig. 22; activation is signed by “+”).

The second effect of norepinephrine, the activation of the ATP-dependent slow

sequestration of calcium by increased levels of cAMP, is not sufficient to

override the ATP-independent calcium release from the endoplasmatic

Discussion 60

reticulum resulting in the observed increase of cytosolic calcium. Addition of

GABA to norepinephrine-treated cells did not change cytosolic calcium as

compared to norepinephrine alone. GABA reduces the cAMP concentration and

causes thereby a reduction in calcium sequestration (Fig. 22; signed by “-”).

This interruption of the previously described calcium cycling [Lang K, et al.,

2002] results in the reduction of locomotor activity.

Figure 22: G protein-mediated calcium cycling. Norepinephrine (NOR) activates Gs protein-coupled heptatransmembrane receptor, which opens calcium ion cannels, sign with “+”, in theresult of fast release of calcium (Ca) from the endoplasmatic reticulum (ER). The same receptoractivates the calcium pump SERCA, which sequestrate calcium into internal stores. TheGi protein-coupled receptors for GABA signalling open the calcium ion cannels, but inhibit (“-“)the calcium sequestration into intracellular stores.

The force of locomotor activity is discussed to be mainly managed by actin

polymerisation and depolymerisation. Our observation of the localisation and

concentration of EGFP-actin in transfected tumour cells show that high actin

concentrations are localised in the subcortical area of the tumour cells body and

in higher amounts at the basis of pseudopods. The shifting of actin in the basis

of the pseudopod is supposed to be the motor force for a rearrangement of the

cell. Interestingly, these findings in our 3D-assay stand in contrast to those of

other using 2D-assays. These 2D-assays show actin as so-called stressfibers

GABA NOR

ER

++

Ca Ca

Gi Gs

--+

Discussion 61

[Choidas A, et al., 1998, Katoh K, et al., 2001]. As Niggemann et al. have

recently shown in a 3D system with fixed MDA-MB-468 colon carcinoma cells

[Niggemann B, et al., 2004] and in our herein presented staining of living cells in

three-dimensional assay, these stressfibers can not observe. Three-

dimensional assays allow a much closer look to what happens in living

organisms and these results of migrating cells in threedimensional assay are

much closer to in vivo conditions [Abbott A, 2003].

Another major point of interest was to see if GABA and anandamide have an

influence on the immune system. We added these substances to T lymphocytes

and NG, which were induced to migrate by SDF-1 of fMLP, respectively. Our

results shown the inhibitory effect of GABA and anandamide on SDF-1-induced

T lymphocytes. Interestingly, with specific agonist to the GABA receptors, like

isoguvacine and baclofen, we could not see any influence on the migration of

SDF-1-induced T lymphocytes. With further experiments by stimulating AC with

forskolin or by addition of db-cAMP, we obtained the same effects on migration

as with tumour cells: the importance of cAMP levels. This shows analogies to

tumour cells: a decrease of cAMP is a sufficient stop signal for the SDF-1-

induced migration, but an increase of cAMP is not a sufficient start signal for the

migration of the T lymphocytes. This supports the view that the inhibitory GABA

effect is predominantly or solely mediated by the regulation of cellular cAMP.

With regard to the receptor subtype, the regulation of cAMP shows that GABA

works through a metabotropic GABA receptor. However, since baclofen failed to

inhibit the norepinephrine-induced migration of T lymphocytes, the involved

metabotropic GABA-receptor must be different from that in tumour cells. A few

studies discuss a novel GABAB-receptor, which differs in one subunit to other

Discussion 62

GABAB-receptors. The expression of these metabotropic GABA receptors in the

central nerve system differs from GABAB-receptors found in peripheral tissue

[Calver A R, et al., 2000, Calver A R, et al., 2003]. This could be an answer to

the uneffectivity of the specific GABAB-receptor agonist baclofen in T cells.

Furthermore, JWH 133, the specific CB2-receptor agonist inhibits the SDF-1-

stimulated migration of T lymphocytes. As discussed above, the norepinephrine

induced tumour cell migration is in contrast inhibited via the CB1-receptor.

The most striking consequence of these results with regard to a possible

therapeutic approach using GABA and cannabinoid receptor agonists in the

inhibition of tumour cell migration is the fact that the inhibitory effect in tumour

cells is mediated via other receptors than in T lymphocytes, and, furthermore,

neither GABA nor anandamide affected NG. Thus, specific agonist for GABAB-

and CB1-receptors such as the endogenously produced DEA [Pertwee R G,

1999] and the therapeutic used baclofen [Tatsuta M, et al., 1990], might be

selective tools for the pharmacological inhibition of metastasis formation, as has

been discussed by Ortega [Ortega A, 2003], without an immunosuppressive

effect, as observed in cannabinoid therapy [De Petrocellis L, et al., 2000]

[Kaminski N E, et al., 1994]. In addition to the herein described effects on

migration, anandamide has been shown to have antiproliferative and apoptosis-

inducing properties in metastatic prostate cancer cells by inducing ceramide

production [Mimeault M, et al., 2003]. Baclofen, the GABAB-receptor agonist,

has therapeutic relevance as myotonolytikum and is discussed, like

anandamide, as a drug for the treatment of multiple sclerosis [Miksa I R,

Poppenga R H, 2003] and more importantly for the reduction of tumour cell

growth [Ortega A, 2003].

Discussion 63

In conclusion, recent findings in cell migration research have shown that not

only the migration of leukocytes is strongly influenced by ligands to serpentine

receptors, i.e. chemokines and neurotransmitters, but the migration of tumour

cells also depends on such signals from the environment. Thus has the concept

of metastasis formation shifted from a solely genetically determined standpoint

to an understanding of the complex interplay of signal substances of the

immune system and the neuro-endocrine system with tumour cells [Entschladen

F, et al., 2002, Seifert P, et al., 2002] (see Fig. 23).

Figure 23: Network of interactions between the neuroendocrine system, the immunesystem, and tumour cells. Beneath the interaction of the three systems other organ systems(signed with a dot) could interact with the triangular network.

Acknowledgements 64

Acknowledgements

A lot of people were crossing my way druing my Ph. D. studies. Everyone of my

colleagues had relevance for me on this way. The most grateful

acknowledgement goes to Prof. Dr. Dr. Kurt Zänker, who made everything

possible in this wonderful institute. Thanks a lot to PD Dr. Frank Entschladen as

a gifted teacher, and Dr. Bernd Niggemann for constructive discussions. I am

grateful to Prof. Dr. Bertram for reviewing this work. Many thanks to the three

MTLAs of this institute: Beate Mainusch, Sylvia Keil and Gaby Troost, for giving

me help and a wonderful working atmosphere. At last, thanks to Dr. Kerstin

Lang for her excellent and productive collaboration and Dr. Ted Drell IV for his

critical reading.

This work was financially supported by the Fritz Bender Foundation (Munich),

and the Bruno and Helene Jöster Foundation (Cologne).

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Curriculum Vitae 72

Curriculum Vitae

Last name : Joseph

Surname : Jan

Date of birth : 07-04-70

Place of birth : Prüm (Eifel), Germany

Gender : Male

Nationality : German

1976 – 1980 Elementary school in Wallersheim

1980 – 1982 Grammar school in Prüm (Regino-Gymnasium)

1982 – 1986 Secondary school in Prüm (Kaiser-Lothar-Realschule)

1986 – 1989 Apprenticeship as a chemical-technician at the Bayer AG in

Leverkusen

1989 – 1991 Civilian service at the town-hospital in Leverkusen

1991 – 1992 Technical college in Leverkusen

July 1992 Advanced technical college certificate

1993 Worked as a chemical-technician at the AGFA-GEVAERT

AG in Leverkusen; Area Production and quality assurance.

1993 – 1998 Studies food chemistry at the “Bergischen Universität

Gesamthochschule Wuppertal”

Nov 1998 State examination first degree

Mai 1999 – Mai

2000

Practical training in food chemistry

July 2000 State examination second degree

Jan 2001 Ph. D. student at the “Institute of Immunology” of the

University in Witten/Herdecke

Jan Joseph

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