Regulation of telomere length and organisation in human skin cells in vitro and in vivo [Elektronische Ressource] / presented by Damir Krunic

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• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: English
• Poids de l'ouvrage: 27 Mo

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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