Comparative cell biological analyses of proto-type galectins in colon cancer [Elektronische Ressource] / von Joachim Christian Manning

Joachim C. Manning

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Aus dem Institut für
Physiologie, Physiologische Chemie und Tierernährung
der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München
Vorstand: Univ.-Prof. Dr. rer. nat. H.-J. Gabius


Angefertigt unter der Leitung von
Dr. Sabine André
Univ.-Prof. Dr. rer. nat. H.-J. Gabius




Comparative Cell Biological Analyses of
Proto-Type Galectins in Colon Cancer





Inaugural-Dissertation

zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München

von
Joachim Christian Manning
San Francisco, CA, U.S.A.
München 2006 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München













Dekan: Univ.-Prof. Dr. E. P. Märtlbauer

Referent: Univ.-Prof. Dr. H.-J. Gabius

Korreferent: Univ.-Prof. Dr. W. Hermanns







Tag der Promotion: 28. Juli 2006











































To my mother,

who would have been proud. Table of Contents

Table of contents
ABBREVIATIONS
1 INTRODUCTION 1
1.1 Glycans and Their Still Underappreciated Role as a Carrier of Information 1
1.2 Translation of the Sugar Code 4
1.3 Galectins 9
1.4 Proto-Type Galectins-1, -2 and -7 13
1.5 Galectins in Cancer 16
2 OBJECTIVES 18
3 MATERIALS, METHODS AND EQUIPMENT 19
3.1 Materials 19
3.2 Equipment 21
3.3 Cell Culture 22
3.3.1 Cell Lines 22
3.3.2 Cell Culture Conditions 22
3.3.3 Passaging of Cells 23
3.3.4 Generation of Stable Cell Transfectants 24
3.3.5 Origin of Cell Clones 25
3.3.6 Counting Cells 26
3.3.7 Freezing Cells 26
3.3.8 Thawing Cells 27
3.4 Biochemical Methods 27
3.4.1 Preparation of Cell Lysates for SDS-PAGE Analysis 27
3.4.2 SDS-PAGE 28
3.4.3 Electrophoretic Transfer of Protein to Nitrocellulose 31
3.4.4 Detection of Galectins on the Blot 32
3.4.5 Enzyme Linked Immunosorbent Assay 34
3.4.6 FACS Analysis 37
3.4.7 Cytochemical Analysis 39
i Table of Contents

3.5 Molecular Biology 39
3.5.1 Isolation of Genomic DNA 39
3.5.2 RNA Extraction, Reverse Transcription of RNA and PCR Analysis 40
3.5.3 Agarose Gel Electrophoresis 41
3.6 Video Microscopy 42
3.7 Assays with Cell Lines 43
3.7.1 Methylcellulose Assay 43
3.7.2 Doubling Time 44
3.7.3 MTT Assay 45
3.7.4 Statistical Analyses 48
4 RESULTS AND DISCUSSION 49
4.1 Characterization of Cell Clones 49
4.1.1 Generation and Selection of Stable Cell Clones 49
4.1.2 DNA and RNA Analysis 49
4.1.3 Analysis of Galectin Protein by ECL Blot 52
4.1.4 Enzyme Linked Immunosorbent Assay 55
4.1.5 FACS Analysis 56
4.1.6 Immunocytochemical Analysis 59
4.1.7 Morphology 66
4.2 Comparative Assays 68
4.2.1 Methylcellulose Assay 68
4.2.2 Doubling Time 71
4.2.3 MTT Assay 77
4.2.4 MTT Assay With Cytotoxic Drugs Added to the Medium 95
5 SUMMARY 103
6 BIBLIOGRAPHY 104
7 DANKSAGUNG 117
8 CURRICULUM VITAE 118
ii Abbreviations

Abbreviations

°C degree Celsius
Ab antibody
approx. approximately
APS ammonium persulfate
Aqua dest. aqua destillata
ASF asialofetuin
BSA bovine serum albumin
CD cluster of differentiation
CRD carbohydrate recognition domain
Cdk cyclin-dependent kinase
DAPI 4'-6-diamidino-2-phenylindole
cDNA complementary deoxyribonucleic acid
DEPC diethylpyrocarbonate
DMSO dimethyl sulfoxide
DPBS Dulbeco’s phosphate-buffered saline
DNA deoxyribonucleic acid
dt doubling time
DTT dithiothreitol
E extinction
EDTA ethylenediaminetetraacetic acid
ELISA enyme-linked immunosorbent assay
ERK extracellular signal-related kinase
EtOH ethanol
FACS fluorescence-activated cell sorting
FCS fetal calf serum
FITC fluorescein isothiocyanate
gal-1 galectin-1
gal-2 galectin-2
gal-7 galectin-7
h hours
iii Abbreviations

IC concentration of toxin required for 50 % inhibition of control value 50
IgG immunoglobulin G
LAD II leukocyte adhesion deficiency syndrome II
LB Luria Bertani
MEK mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
min minutes
mPa s millipascal second
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
No. number
OD optical density
OPD o-phenylenediamine dihydrochloride
p p-value
PAGE polyacrylamide gel electrophoresis
PBS phosphate-buffered saline (20 mM, pH 7.2)
PCR polymerase chain reaction
rpm rounds per minute
sec seconds
SD standard deviation
SDS sodium dodecyl sulphate
SN38 7-ethyl-10-hydroxy-20(S)-camptothecin
TAE Tris-acetate-EDTA buffer
TE buffer Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer
TEMED N, N, N', N'-Tetramethylethylenediamine
TBS Tris(hydroxymethyl)aminomethane-buffered saline
Tris Tris(hydroxymethyl)aminomethane
T-TBS Tris(hydroxymethyl)aminomethane-buffered saline with 0.05 % v/v Tween-20
V Volt
x “x” refers to the fold concentration of the stock solution
x g times gravity (in context with centrifuge speed)
WT wild-type

iv Introduction

1 Introduction
Living organisms are made up of one or more cells, can grow, reproduce, process
1information, carry out chemical reactions, and respond to stimuli . Information transfer is a
necessary precedent to responsiveness of cells to stimuli in their environment. The route of
information transfer in living organisms cannot be a one-way street, though. Coordination in
multicellular organisms requires feedback mechanisms between cells and to the environment.
This must allow cells to respond to different challenges swiftly and accurately.
There is no doubt to the role of nucleic acids and amino acids in coding information in form
of DNA, RNA and protein. However, it is becoming clearer that the whole story is not told,
when describing the genome and proteome as the only hardware coding information of living
2, 3organisms. In the concept of the genetic code, a new concept is emerging: the sugar code .
This describes glycans as a third class of bio-informative macromolecules, next to nucleic
acids and proteins. As a first step, the complexity of all glycans being produced by an
4organism, the glycome, is being identified . In the past, the lack of possibilities to analyze
glycan chains in detail was a main reason why scientists did not pay adequate attention to the
potential information-encoding system hidden in sugar structures. Today, sophisticated
5, 6, 7analytical procedures are at hand and thus these problems have been elegantly mastered .
Therefore in the next chapter, the view can be focused on saccharides as possible coding
units.
1.1 Glycans and Their Still Underappreciated Role
as a Carrier of Information
Carbohydrates are well known as carriers of energy, such as starch and glycogen, and as main
structural elements in plants and insects, such as cellulose and chitin. One noteworthy
common characteristic of these four examples is, as very different as their form and function
may be, that all are polysaccharides made up of repeating units of glucose, connected via the
C1 and C4 carbon atoms. Differences between the anomeric linkage (α-D-glucose in starch
and glycogen, or β-D-glucose in cellulose) or N-acetylation of C2 (β-D-N-acetylglucosamine
in chitin) results in the diversity of the final structures.
1 Introduction

Nucleic acids and amino acids encode information by being linked to each other in a specific
order. Each single element has enough structural diversity from the other elements to be
distinct. The question as to whether sugars also meet these requirements will now be
addressed. The here presented reasoning to the importance of sugars follows the guidelines
3provided by Gabius et al. . These authors emphasize the meaning and validity of the sugar
code as an information carrier. The pyranose/furanose ring of monosaccharides presents many
hydroxyl groups suitable for donor/acceptor bonds or for coordination bonds with cation such
2+as Ca . The sugar D-galactose has a set of weakly polarized C-H bonds, one side of
C-H/π-electron and van der Waals interactions. This makes stacking to aromatic ring systems
possible, e.g. to the indolyl ring of tryptophan. The sugar D-galactose can also be used to
exemplify the consequences of changes in positioning of a hydroxyl group in epimers. As in
other monosaccharides, shifting the hydroxyl group to the other side of the ring will affect the
potential for directional hydrogen bonds. An additional consequence will b