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Dissertation


submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences


Regulation of Telomere Length and Organisation in
Human Skin Cells in vitro and in vivo












presented by
Graduated Engineer of Biology Damir Krunic
2008




Dissertation


submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Science







Presented by
Graduated Engineer of Biology Damir Krunic
Born in: Slavonski Brod, Croatia
Oral-examination: ......................

2008






Regulation of Telomere Length and Organisation in
Human Skin Cells in vitro and in vivo








Referees: Prof. Dr. Thomas Efferth
Prof. Dr. Petra Boukamp




The present doctoral thesis has been carried out in the Department “Genetics of
Skin Carcinogenesis” at the German Cancer Research Center, Heidelberg, under
supervision of Prof. Dr. Petra Boukamp.













Herewith, I declare that I am the sole author of the submitted dissertation and
made no use of any sources or help apart from those specifically referred to.




(Date) (Signature)
Acknowledgements
Acknowledgements

There are too many people I would like to acknowledge, without their help and
support this thesis would never come to exist.
First of all, I would like to thank to Prof. Dr. Thomas Efferth for supervising my thesis
at the Ruperto Carola University of Heidelberg.
I especially want to thank to Prof. Dr. Petra Boukamp for being a great supervisor at
the German Cancer Research Centre. It is not always possible to find a position in a good
group that is working exactly on the subject you are interested in; thank to the fact that Petra
gave me her trust - it was possible for me.
Each good group is, of course made out of good members, and I would like to thank
those present and past, colleagues and friends, for their help and advice, as well as for the
good working atmosphere that never lacked in the Boukamp´s lab, namely; to Sharareh
Moshir for the help in my first steps in the lab, to Karin Greulich-Bode for being a great
FISHing guide and proof-reader of the thesis, to Hans-Jürgen Stark for the help with immune
staining and a healthy humour, to Hermann Stammer, Katrin Schmidt and Iris Martin for very
professional technical assistance. Further thanks to Mara Amoros, Berit Falkowska-Hansen,
Katrin Wischerman, Karston Böhnke, Sonja Muffler, Felix Bub, Sybille Ermler, Sabine
Rosenberger, Susanne Buschke, Jutta Laykauf, Christine Baderschneider, Sabrina
Gundermann, Pavle Krsmanovic, Petra Sanders, and Susanne Popp. Thanks also to Christa for
beautiful immune stainings, to Melanie and to Dirk, and to Müller and Franke group for being
good helping neighbours on the floor.
Special thanks to my colleagues and friends from the university in Croatia that were
PhD students at DKFZ and EMBL, namely Alen, Josipa, Vibor, Ana, Moki, Kreso, Filip, and
there associates; Andreas, Timo, Dilem, and to Jean, Zoran, Snjezana and others that were
very helpful from the beginning, and made my living abroad much easier. Fruitful exchange
of experience from our two institutes was often subject of our meetings in Untere Straße.
Finally, biggest thanks to my wife, first of all for deciding to became one, although she
was fully aware of all the good and bad sides of the scientific career. She was and still is a
great support, like she promised – in good and bad. Thanks also to my family, parents,
brothers and friends in Croatia – it is always nice to know that even if no experiment works,
and if everything goes wrong, at least vacation with all of them will be great.

















































To Rko,
mynewbrn son.




Abstract
Abstract
Telomeres are specialized DNA-protein structures at the ends of the linear chromosomes that form
protective caps. They are composed of multi-fold double-stranded 5’-TTAGGG-3’ repeats and a 3’
single stranded overhang that loops back and invades the duplex region. The so called T-loop structure
is stabilized by a number of associated proteins that protect the DNA against degradation and hinders
the cellular machinery to recognize the ends as broken DNA, thus being essential for chromosomal
integrity. Investigating the three-dimensional (3D) telomere distribution we now show that telomeres
of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the
inner third of the nuclear space in interphase cells and extend more widely during mitosis. This
distinct localization is abrogated in a HaCaT variant that constitutively expresses the c-Myc onco-
protein. Telomeres in HaCaT-myc cells form aggregates (TAs) that are accompanied by an overall
irregular telomere distribution in interphase. Since this TA formation also leads to clustering of the
respective chromosomes and TA formation is present during mitosis, TAs most likely contribute to
genomic instability by forcing abnormal chromosome segregation. As a first step to approach the
mechanism of TA formation we compared the difference in nuclear protein expression between
HaCaT and HaCaT-myc cells by two-dimensional polyacrylamide gel electrophoresis. Out of 30
differentially expressed proteins, the most promising candidate was Matrin 3, a nuclear matrix protein
that, being reduced in HaCaT-myc cells, suggests for a mechanism involving incorrect adhesion to the
nuclear matrix.
The most essential function of the telomeres is their role as protective caps and thus their role in
guaranteeing chromosomal integrity. Telomeres shorten with each replication cycle (end-replication-
problem) and accordingly, telomere shortening is supposed to be a major cause of aging in
proliferatively active tissues. To investigate the role of telomere loss for skin aging in situ, we
developed a 3D deconvolution microscopy based Q-FISH/immunofluorescence technique on
individual cells in tissue sections. When investigating skin from different-age donors, we found that
similar as for dermal fibroblasts and another non-proliferative cell type in the epidermis, the
melanocytes, also the epidermal keratinocytes only show a minimal age-dependent telomere decline.
Thus age-dependent telomere loss appears largely neglectable. However, we found significant inter-
personal differences and most strikingly, intra-personal variations in telomere lengths between similar
sites of the epidermis. Moreover, in 10 of 30 samples of normal skin, preferentially from sun exposed
sites of elderly donors, we identified regions within otherwise normal looking epidermis with
significantly shorter telomeres. Though size and number of these micro-lesions as well as amount of
telomere shortening varied, all enclosed various basal and suprabasal differentiated cells. Most
importantly, they were all characterized by expression of the p53 tumour suppressor gene and 53BP1
foci co-localizing with telomeres. Since the latter are representative for DNA double strand breaks and
when co-localized with telomeres represent critically short uncapped telomeres, these date
demonstrate that in these micro-lesions the telomeres are dysfunctional and likely represent stages of
genomic instability. Such distinct areas can only be maintained when damage has occurred in a stem
cell. We, therefore, postulate that damage did not cause cell death but was repaired and lead to a
decreased telomere length. This reduced telomere length was then transmitted to the daughter cells.
Thus each micro-lesions most likely represents the space of one stem cell compartment.
We also identified shorter telomeres in skin from heavily sun-exposed individuals and in several
skin sections in sites closer to the surface (outside) as compared to more protected areas of the
epidermis (deeper parts of the rete ridges, deeper parts of the hair follicles). Since we further show that
in skin from 16 volunteers irradiated with UVA, UVB, or a combination of UVA and UVB, distinct
telomere shortening was visible already 3 days post irradiation, UV radiation is clearly a responsible
factor for accelerated telomere loss in human skin. Another factor may be forced oxidative damage
because also chronic and to a lesser extend acute wounds showed distinct telomere shortening.
Finally, we demonstrate for the first time a population of rare cells within the epidermis which have
the “longest” telomeres and which likely represent the stem cells. These cells are distributed rather
randomly throughout the basal layer. Furthermore, when cultured and replated in organotypic cultures
they re-establish as stem cells in the newly developing epidermis. Thus, telomere length is a valuable
marker for stem cells and it is damage rather than replication-dependent telomere shortening that leads
to significant shortening and potential sites of genomic instability in human skin.

Zusammenfassung
Zusammenfassung
Telomere sind spezialisierte DNA-Proteinstrukturen an den Enden der linearen Chromosomen, die
aufgrund ihrer Länge und Struktur als schützende Endkappen die Chromosomenstabilität garantieren. In
der vorliegenden Arbeit konnte nun gezeigt werden, dass in adherent wachsenden Zellen (Keratinotzyten
und Fibroblasten der Haut) die Telomere in allen Phasen des Zellzyklus eine ganz spezifische Verteilung
aufweisen. Die damit verglichenen HaCaT-myc Zellen, die aufgrund der konstitutiven Expression des c-
Myc Onkogens sog. Telomer Aggregate (TAs) enthalten, zeigen dagegen eine generell veränderte
Verteilung. Besonders bemerkenswert war, dass die TAs auch während der Mitose erhalten blieben und in
Anaphase Zellen meist nur in einer der Tochterzellen nachweisbar waren oder dass das gesamte TA in
Mikrokernen ausgeschleust wurde. Diese Befunde deuten darauf hin, dass TAs zu chromosomaler
Missverteilung führen und damit ein möglicher Grund für die Entstehung numerischer Aberrationen sind.
In einem ersten Schritt den Mechanismus zu ermitteln, der zur TA Bildung führt, haben wir die
Kernproteine der HaCaT und HaCaT-myc Zellen mit Hilfe der zweidimensionalen Polyacrylamidgel
Elektrophorese verglichen. Von 30 unterschiedlich exprimierten Proteinen war das Zellmatrixprotein
Matrin 3, der aussichtsreichste Kandidat, da es in den HaCaT-myc Zellen deutlich reduziert war und dies
auf eine inkorrekte Adhesion der Telomere an die nukleäre Matrix hinweist.
Wie die Telomerorganisation so spielt auch die Telomerlänge für die chromosomale Stabilität eine
wesentliche Rolle. Telomere werden mit jedem Replikationszyklus kürzer (Endreplikationsproblem) und
deshalb geht man davon aus, dass Telomerverkürzung die Hauptursache für die Alterung von proliferativ
aktiven Geweben ist. Zur Untersuchung der Rolle des Telomerverlustes bei der Alterung der Haut in situ,
haben wir eine 3D Deconvolutionsmikroskopie-basierte Q-FISH/immunfluoreszenz Technik entwickelt, die
es erlaubt, die verschiedenen Zellen zu identifizieren und ihre Telomere individuell zu analysieren. Mit
dieser Methode können wir nun zeigen, dass in der Haut von Spendern aus verschiedenen Altersgruppen
die Telomere der dermalen Fibroblasten (teilen sich nur selten) aber gleichermaßen auch die Telomere der
ständig proliferierenden epidermalen Keratinozyten eine nur minimale Verminderung der Telomerlänge
zeigen. Somit scheint ein Alters-abhängiger Telomerverlust als Grund für die Hautalterung weitgehend
vernachlässigbar. Dagegen zeigten unsere Untersuchungen aber signifikante Unterschiede zwischen
Spendern gleichen Alters und unerwarteter Weise auch innerhalb eines Spenders. Besonders auffällig
waren „Mikroläsionen“, Areale mit Zellen mit extrem kurzen Telomeren umgeben von Zellen mit normaler
Telomerlänge. Interessanterweise waren diese Bereiche auch durch Expression des Tumorsuppressorgens
p53 und durch 53BP1 Fozi charakterisiert, die mit den Telomeren ko-lokalisierten. Letzteres ist wiederum
charakteristisch für ungeschützte Telomere (repräsentieren nun Doppelstrangbrüche), und weist auf stark
geschädigte Zellen hin, die höchstwahrscheinlich aufgrund kritisch kurzer Telomere genomisch instabil
geworden sind und so eine mögliche erst Vorstufen von Hautkrebs darstellen.
Da diese Bereiche häufig in Haut von stark Sonnen-exponierten Arealen nachweisbar waren, haben wir
dann gezielt Haut nach definierter UV-Exposition untersucht: Hierbei zeigte sich, dass, wenn gleichzeitig
mit UVA und UVB bestrahlt wurde, in der Tat bereits nach 3 Tagen eine deutliche Telomerverkürzung
vorlag,. UVA oder UVB alleine waren dagegen deutlich weniger effektiv. D.h. UV Strahlung scheint ein
wesentlicher Faktor für vorschnelle Telomerverkürzung in der Haut zu sein. Für diese Interpretation
sprechen auch Befunde, dass die Telomere in Bereichen, die weiter entfernt von der Oberfläche lagen (im
unteren Teil der Rete Ridges, oder im unteren Teil der Haarfollikel) und damit gegenüber Strahlung besser
geschützt waren, deutlich länger waren. Ein weiterer Faktor für vorschnelle Telomerverkürzung könnte
massive oxidative Schädigung sein. So fanden wir in chronischen Wunden eine massive Telomer-
verkürzung, während akute Wunden eine deutlich geringere Verkürzung aufwiesen.
Obwohl die Größe der Mikroläsionen als auch der Umfang der Telomereverkürzungen variierte, waren in
allen Fällen die basalen wie auch die suprabasalen, differenzierenden Zellen betroffen. Dies ist nur
möglich, wenn die Schädigung (Telomerbruch) in einer Stammzelle erfolgt ist und diese die verkürzten
Telomere nun an alle Tochterzellen weitergibt. Somit stellt jede Mikroläsion wahrscheinlich ein
Stammzellkompartiment dar. Um die Rolle der Telomere in den Stammzellen noch weiter zu
charakterisieren, haben wir schließlich die Basalschicht der Epidermis im Detail untersucht. Diese Studien
zeigen nun zum ersten Mal, dass es einzelne Zellen gibt, die längere Telomere aufweisen und
wahrscheinlich Stammzellen sind. Diese Zellen liegen „zufällig“ in der Basalschicht verstreut. Nach
Kultivierung und Wiedereinsetzung in organotypische. Kulturen reetablieren sie sich als Stammzellen auch
in der neu sich bildenden Epidermis. Damit ist die Telomerlänge ein neuer wertvoller Marker für die
Identifizierung der Stammzellen und es ist ihre exogene Schädigung und nicht die Replikation, die zu einer
signifikanten Telomerverkürzung und damit zu potentieller genomischer Instabilität in den humanen
Keratinozyten führen kann.

List of abbreviations
List of abbreviations

% Percent MEM Modified Eagle’s minimal
°C Celsius degree essential medium
2+µg, µm, Micro-gram, -meter Mg Magnesium ions
µl, µM Micro -litter, -molar min Minute
3D OTC Three dimensional ml, mM milli-litres, -molar
organotypic co-cultures Na Sodium
3D Three-dimensional NaCl chloride
A Adenine NaOH hydroxide
a.u. Arbitrary units ng, nm Nano-gramm, -meter
A Absorbance on Λ =260 nm No. Number of 260
− ALT Alternative lengthening of ONOO Peroxynitrite
telomeres PBS Phosphate buffered saline
bp base pairs PD Population doubling
BrDu Bromo-deoxyuridine doublings
pH Measure of the acidity or C Cytosine
CA IX Carbonic anhydrase IX alkalinity of a solution
CCD Charge-coupled device RNA Ribonucleic acid
CO Carbon dioxide RNase Ribonuclease 2
CPD Cyclobutane pyrimidine RNP Ribonucleoprotein
dimer ROS Reactive oxygen species
CRBCs CPD retaining basal cells rpm Rotation per minute
Cy2 Cyanine fluorochrome RR Rete ridges
Cy3 Indocarbocyanin fluorochrome rt Room temperature
Da Dalton (molecular size U) SCC Squamous cell carcinoma
DAPI 4´-6-Diamidino-2-phenylindol SDS Sodium dodecyl sulphate
ddHO Double-distilled water SI Signal intensity 2
DMEM Dulbecco’s modified Eagle’s T Thymine
medium TA Telomeric aggregate
DNA Deoxyribonucleic acid TACs Transit amplifying cells
DSB Double strand break TL ere length
EDTA Ethylene-diamine-tetra-acetat T-loop Telomeric loop
FACS Fluorescence activated cell TRF1,2 Telomeric repeat binding
sorting factor 1, 2
FCS Foetal calf serum TRFL Terminal restriction
Fig. Figure fragment length
FISH Fluorescence in situ Tris Tris-(hydroxymethyl)-
hybridization aminomethan
FITC Fluorescein isothiocyanate Triton X-100 Ocylphenol-
g Gram polythylenglycol ether
G Guanine TSI Telomere signal intensity
h Hour TTS Telomere territories
HaCaT Human adult calcium Tween 20 Polyoxyethylen-
temperature (keratinocyte cell sorbitanmonolaurat line) U Unit
HaCaT-myc myc transfected HaCaT UV Ultraviolet light
HCl Hydrochloric acid V Volt
HF Hair follicle vs. Versus
hTERT Human telomerase catalytic α Level of significance
subunit Λ Wave length in nm
hTR Human telomerase RNA Q-FISH Quantitative fluorescence
ID Identification number in situ hybridization
IUdR Iododeoxyuridine
IFE Interfollicular epidermis
kb Kilobase
LRC Label retaining cell
M, N Molar, normal
mA, mg, mm Milli-ampere, -gramm, -meter
MCSP Melanoma associated
chondroitin sulphate
proteoglycane





Table of contents
TABLE OF CONTENTS
1 INTRODUCTION...................................................................................................1
1.1 THE TELOMERE STRUCTURE ................................................................................. 2
1.1.1 The Telomere sequence............................................................................................................ 2
1.1.2 Telomeric proteins.................................................................................................................... 3
1.1.3 Higher order telomere structure................................................................................................ 4
1.2 THREE-DIMENSIONAL DISTRIBUTION OF TELOMERES IN THE NUCLEUS .............. 6
1.2.1 Telomere interactions in the nucleus........................................................................................ 7
1.2.1.1 Telomere clustering in meiosis............................................................................................................... 7
1.2.1.2 Telomeric associations in normal and tumour cells............................................................................... 7
1.2.1.3 aggregates............................................................................................................................. 8
1.3 TELOMERE LENGTH REGULATION......................................................................... 9
1.3.1 Telomere shortening in vitro .............................................................................................................. 9
1.3.2 length regulation in vivo ................................................................................................... 10
1.3.2.1 Telomerase activity in somatic cells..................................................................................................... 11
1.3.2.2 Telomeres and Telomerase in immortalized cell lines and tumours..................................................... 12
1.4 TELOMERES IN HUMAN SKIN................................................................................ 13
1.4.1 Constant proliferation in epidermis requires telomere length maintenance by telomerase .... 13
1.4.2 Telomere length in the skin.. 14
1.4.3 Damage on telomeres in the skin............................................................................................ 15
1.4.3.1 UV damage on telomeres ..................................................................................................................... 15
1.4.3.2 Chronic inflammation and damage on telomeres................................................................................. 16
1.4.4 p53 signalling and damage in the skin.................................................................................... 17
1.4.5 Search for stem cells in the human skin. 18
1.4.5.1 Label retaining cells in three-dimensional organotypic cocultures (3D-OTCs) .................................. 19
1.4.5.2 Melanoma chondroitin sulphate proteoglycan (MCSP) and carbonic anhydrase IX (CA-IX) as a
potentional stem cell markers .................................................................................................................. 19
1.4.5.3 Telomere length in stem cells ............................................................................................................... 20
1.5 OBJECTIVES.......................................................................................................... 22
2 MATERIALS AND METHODS...........................................................................23
2.1 MATERIALS. 24
2.1.1 List of Chemicals.................................................................................................................... 24
2.1.2 DNA and PNA probes, DNA-counterstains and length-markers ........................................... 25
2.1.3 Enzymes and antibodies ......................................................................................................... 26

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