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Involvement of the lysosomal cysteine peptidase cathepsin B in tumour progression and formation of metastases [Elektronische Ressource] / vorgelegt von Anna Papazoglou

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168 pages
INVOLVEMENT OF THE LYSOSOMAL CYSTEINE PEPTIDASE CATHEPSIN B IN PEPTIDASE CATHEPSIN B IN TUMOUR PROGRESSION AND FORMATION OF METASTASES Inauguraldissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität, Freiburg i. Br., Deutschland Vorgelegt von Anna Papazoglou aus Athen, Griechenland November 2003 Die vorliegende Arbeit entstand im Institut für Molekulare Medizin und Zellforschung der Albert-Ludwigs-Universität, Freiburg i. Br., Deutschland, unter der Anleitung von Prof. Dr. C. Peters. Dekan: …………………………………….. Promotionsvorsitzender: Betreuer der Arbeit: Prof. Dr. Christoph Peters Weiterer Betreuer: …………………………………….. Referent: Prof. Dr. Christoph Peters Ko-Referent: Prof. Neubüser ii I will lift my eyes to the hills wher my help comes from? My help comes from the LORD, who made heaven and earth. Psalm 121;1-2 iii ACKNOWLEDGMENTS I am telling everyone that this thesis was, entirely, the product of my own hard labour, but this is not really true. Throughout this work several people have offered their contribution and they did so in many different ways. So, to all those who have helped, I want to express my most sincere thanks. However, some people deserve a special mention.
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INVOLVEMENT OF THE
LYSOSOMAL CYSTEINE
PEPTIDASE CATHEPSIN B IN PEPTIDASE CATHEPSIN B IN
TUMOUR PROGRESSION AND
FORMATION OF METASTASES















Inauguraldissertation zur Erlangung der Doktorwürde der Fakultät für
Biologie der Albert-Ludwigs-Universität, Freiburg i. Br., Deutschland





Vorgelegt von
Anna Papazoglou
aus Athen, Griechenland

November 2003















Die vorliegende Arbeit entstand im Institut für Molekulare Medizin und
Zellforschung der Albert-Ludwigs-Universität, Freiburg i. Br.,
Deutschland, unter der Anleitung von Prof. Dr. C. Peters.




Dekan: ……………………………………..
Promotionsvorsitzender:
Betreuer der Arbeit: Prof. Dr. Christoph Peters
Weiterer Betreuer: ……………………………………..
Referent: Prof. Dr. Christoph Peters
Ko-Referent: Prof. Neubüser
ii


























I will lift my eyes to the hills
wher my help comes from?
My help comes from the LORD,
who made heaven and earth.

Psalm 121;1-2
iii














ACKNOWLEDGMENTS


I am telling everyone that this thesis was, entirely, the product of my own hard labour,
but this is not really true. Throughout this work several people have offered their
contribution and they did so in many different ways. So, to all those who have helped, I
want to express my most sincere thanks.
However, some people deserve a special mention. First of all, I have to thank Professor
Christoph Peters the director of the Institute of Molecular Medicine and Cell Research for
suggesting a project with so many possibilities to explore, and for taking me on in the
first place. Wera Roth has guided my fist steps as a PhD student. Jan Deussing, my
supervisor, has helped immeasurably with his scientific contributions as well as with his
friendship. I would like also to thank Thomas Reinheckel, for his constructive criticism
and methodological advice on my thesis manuscript.
My thanks to Ingrid Werber; Ingrid working with you side-by-side during the last four
years was a privilege. Thanks for being you. I would like also to thank the rest of the
incredible AG Ex-Deussing for their laboratory assistance, patience, and understanding
while they were working with me in the lab. All of you guys have created a great working
atmosphere.
I would also like to thank Michael Stock, Mateusz Kolanczyk and the rest of the lab mafia.
You were my partners in crime. Going for drinking and dancing with you was always a
pleasure!!!! Michael, I think you still owe me a bottle of wine. Mateuz I think it is time to
go dancing again.
My greatest thanks are reserved for those who encouraged, continuously supported and
helped me the most, my parents and my brother. Their belief in my abilities and me
provided the encouragement to achieve my goals and go far beyond my expectations.
For this reason and their love I owe them a debt of thanks. Dad thanks for the thesis
iv Acknowledgments


manuscript proof reading. It was a great help. Mom your prayers are always following me
wherever I am, thanks!!!

A big thanks goes to Alexandros for being always there for me. You are a good friend.

There are several more people I would like to thank. AG Nikkhah, I would like to thank
you all for being patient and understanding. I tried hard to balance my life between you
and my thesis during the last year. I hope I have not disappointed you. Thank you for
trusting me and being there for me. Professor Nikkhah a great thanks for having
unlimited patient and understanding. Thank you for believing in me ………………………

Above all, I would like to thank GOD. He did not only give me the opportunity to come
and study in Germany but He was always there next to me, supporting me and
encouraging me through my PhD. Whatever I am and whatever I manage to become in
life it is because of HIM. He is the ALPHA and the OMEGA, the beginning and the end of
my life!


v













ABSTRACT


The leading cause of cancer deaths is tumour metastasis. The formation of metastases is a
complex multistep process, involving invasion of the basal membranes and connective
tissue, penetration of the blood vessel walls, invasion of the host tissue, and initiation of
growth at the new location. The degradation of extracellular matrix and basal membranes by
proteolytic enzymes makes possible the invasive growth of tumour cells. Numerous reports
describe increased levels of proteolytic enzymes in invasive tumours. These proteases
include neutral metalloproteinases, serine proteinases as well as lysosomal, aspartic and
cysteine peptidases.
To investigate the role of the lysosomal cysteine peptidase cathepsin B (CTSB) in tumour
progression and metastasis, transgenic mice FVB/N-TgN(MMTVPyVT)634Mul, developing
multifocal metastasising mammary carcinomas, were utilised. CTSB deficient mice, with a
mixed C57BL/6J and 129SvJ background were made congenic for the FVB/N strain. FVB/N-
TgN(MMTVPyVT)-CTSB, wt, ht and ko were generated through inter-crossing (PyCB strain).
The mice were sacrificed sixty days after the discovery of the first palpable mammary
tumour. The weight of primary tumours was measured and the volume of the pulmonary
metastases was evaluated by histomorphometry.
The genotype was found to be an influential factor on the primary tumour weight as well as
on the total metastasis volume in the lungs but played no role on the timing of the initiation of
the primary mammary tumour. Statistical analysis showed that the adjusted mean of primary
vi Abstract


+/+ +/-tumour weight for the Py;Ctsb (n=36) is significantly higher compared to Py;Ctsb (n=38,
-/-p=0.006) and Py;Ctsb (n=32, p=0.001). Surprisingly, in the case of the total lung metastasis
+/+ -/-volume, the adjusted means of the genotypes Py;Ctsb and Py;Ctsb are not significantly
+/+ +/-different (p=0.13) but there is a significant difference between Py; Ctsb andPy; Ctsb
(p=0.01).
Histological evaluation of the PyCB female mice, of the three different genotypes, shows no
difference in tumour development and extravasation as well as in lung metastasis
development.
In order to study the formation of colonisation and metastasis, independently from the
5primary tumour formation, 5 x 10 primary tumour cells of different CTSB genotypes were i.v.
injected into FVB/N congenic females of different CTSB genotypes. The animals were
sacrificed two weeks after the injection and the lung metastases were evaluated. The total
+/+ volumes of the metastases were significantly smaller, when ctsb tumour cells were injected
-/- +/- +/+ -/- 3 +/-into ctsb females, compared to ctsb or ctsb recipients (ctsb 0.92 mm ( ± 0.42), ctsb
3 +/+ 3 2.37 mm ( ± 1.09), p= 0.0013; ctsb 2.16 mm ( ± 0.99), p=0.006). No differences in the total
-/-metastasis volume were observed with the injection of ctsb tumour cells, compared to
+/+ctsb .
In situ hybridisation of mammary tumour tissue and lung metastatic tissue of FVB/N-
TgN(MMTVPyVT)634Mul female mice showed a high expression of cathepsin B and
cathepsin L in the area of the tumour. In the case of the lung metastases the expression of
both cathepsins was concentrated on the borders of the metastasis suggesting that the cells
that surround the metastasis might produce CTSB and CTSL. Furthermore,
immunohistological analysis of the lung metastatic tissue showed that most of these cells
were macrophages, as staining with the macrophage specific antibody F4/80 confirmed.
Double immunofluorescence revealed that these tumour-surrounding macrophages express
high levels of CTSB.
These data indicate that extracellular CTSB produced by inflammatory stromal macrophages
plays a role in primary tumour growth as well as in metastasis formation and growth. CTSB
+/- -/-expression enhances tumour growth. However, Py;ctsb and Py;ctsb tumour cells
+/+ -/-proliferate less compared to Py;ctsb but Py;ctsb tumour cells have a higher ability to
+/+ +/- degrade extracellular matrix proteins than Py;ctsb and Py;ctsb tumour cells.
vii













ABBREVIATIONS


% Percentage
Microgram µg
Microlitre µl
µm Micrometer
0C Degrees of Celsius
bFGF Basic Fibroblast Growth Factor
bp Base Pairs
BSA Bovine Serum Albumin
C Concentration
cDNA Complementary DNA
2cm Square Centimetre
CO Carbon dioxide 2
cpm Counts per minute
-/- Cathepsin B deficient genotype ctbl
CTP Cytidin triphosphate
CTSB Cathepsin B
+/-ctsb Cathepsin B heterozygous genotype
+/+ctsb Cathepsin B wild type
CTSL Cathepsin L
-/-ctsl deficient genotype
+/-ctsl heterozygous genotype
+/+ctsl Cathepsin L wild type
dATP Deoxyadenosin triphosphate
dCTP Deoxycytidin triphosphate
DDT Dithiothreitol
dGTP Deoxyguanidin triphosphate
DMEM Dubecco’s Modified Eagle Medium
DMSO Dimethyl Sulphoxide
viii Abbreviations


DNA Deoxyribonucleic acid
DNAse Deoxyribonuclease
dNTP Deoxynucleotide triphosphate
DTT Dithiothreitol
dTTP Deoxythymidin triphosphate
EDTA Ethylene Diamine Tetraacetate
EGF Epidermal Growth Factor
ER Endoplasmatic Reticulum
et al. et alii (lat: and others)
FCS Fetal Calf Serum
g Gram
GEM Genetic Engineered Mice
h Hour
HO Water 2
HCl Hydrochloric acid
ht Heterozygous
HT-1080 Human fibrosarcoma
IL-2 Interleukin–2 gene
kb Kilobase
kDa Kilodalton
ko Knock out (deficient)
LTR Long Terminal Repeat
M Molarity
M6P Mannose 6-phosphate
mg Milligram
min Minute
ml Millilitre
mm Millimetre
MMPs Matrix Metalloproteinases
MMTV Mouse Mammary Tumour Virus
MOPS [3,[N-morpholino]-propanesulfonic acid]
mRNA Messenger Ribonucleic Acid
MT Middle T
NaOAC Sodium Acetate
NaOH Sodium Hydroxide
NCBI National Centre for Biotechnology Information
NCI National Cancer Institute
NIH-3T3 Murine fibroblast
ns not significant
nm Nanometre
OD Optical Density
PBS Phosphate Buffer Saline
PCR Polymerase Chain Reaction
PDB Protein Data Bank
PFA Paraformaldehyde
PyVT Polyoma Virus Middle T
uPA Urokinase Plasminogen Activator
RER Rough Endoplasmic Reticulum
RNA Ribonucleic Acid
RNase Ribonuclease
rpm Revolutions per minute
RT Room Temperature
RT-PCR Reverse Transcriptase PCR
s Second
ix Abbreviations


SDS Sodium Dodecyl Sulphate
SE Standard Error
SV40 Simian Virus
T-Ag T antigen
TBE Tris-Borate EDTA buffer
TGF- β Transforming Growth Factor β
Tumour Necrosis Factor Alfa TNF- α
Tris Tris(hydroxymethyl)-aminoethan
TRY Trypsinogen
TSG Tumour Suppressor Gene
U Units
UV Ultraviolet
V Volt
v/v Volume per Volume
w/v Weight per Volume
wt Wildtype


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