dc.contributor.author
Finzel Pérez, Ana
dc.date.accessioned
2018-06-07T20:54:50Z
dc.date.available
2016-12-19T09:19:39.701Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/7115
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-11314
dc.description
1 INTRODUCTION
........................................................................................................................................
1 1.1 P53 IS A CELLULAR GATEKEEPER
............................................................................................................
1 1.1.1 Functional domains of p53
................................................................................................................
1 1.1.2 p53 is activated upon stress
..............................................................................................................
2 1.2 RESPONSE TO DOUBLE STRAND BREAKS
.................................................................................................
3 1.2.1 Homologous recombination
..............................................................................................................
4 1.2.2 Canonical non-homologous end joining
...........................................................................................
5 1.2.3 p53 is activated upon genotoxic stress
..............................................................................................
6 1.3 IMPORTANCE OF P53 SINGLE CELL STUDIES
............................................................................................
8 1.4 P53 AND METABOLISM
............................................................................................................................
9 1.5 AIMS OF THE STUDY
.............................................................................................................................
12 2 MATERIALS AND METHODS
...............................................................................................................
13 2.1 CELL CULTURE
.....................................................................................................................................
13 2.2 INHIBITOR AND RADIATION
TREATMENTS.............................................................................................
14 2.3 TIME-LAPSE MICROSCOPY
....................................................................................................................
14 2.4 IMAGE ANALYSIS
..................................................................................................................................
15 2.5 WESTERN BLOT ANALYSIS
....................................................................................................................
16 2.6 RT-QPCR
.............................................................................................................................................
18 2.7 CELL PROLIFERATION ASSAY
................................................................................................................
20 2.8
IMMUNOFLUORESCENCE.......................................................................................................................
20 2.9 SIRNA TREATMENT
..............................................................................................................................
21 2.10 RNA-SEQ EXPERIMENT WORKFLOW
.....................................................................................................
22 2.11 RNA-SEQ DATA ANALYSIS
...................................................................................................................
26 2.11.1 Calculation of the distance score
................................................................................................
27 2.11.2 Functional annotation analysis
...................................................................................................
27 2.12 PROTEOMICS
........................................................................................................................................
28 2.12.1 Data analysis
..............................................................................................................................
30 3 RESULTS
....................................................................................................................................................
31 3.1 UPON GENOTOXIC STRESS, P53 CONTROLS ENERGY PRODUCTION THROUGH
MITOCHONDRIAL RESPIRATION AND PROMOTES EXPRESSION OF KALLIKREINS
.............................................................................
31 3.1.1 Analysis of the p53 transcriptional response under basal conditions
and upon genotoxic stress ... 31 3.1.2 Combination of RNA sequencing and
proteomics data points to a role of p53 in controlling energy production upon
irradiation
.........................................................................................................................
36 3.1.3 Kallikrein expression is p53-dependent
..........................................................................................
42 3.2 HYPER-ACTIVATION OF ATM UPON DNA-PKCS INHIBITION MODULATES P53 DYNAMICS
AND CELL FATE IN RESPONSE TO DNA DAMAGE
................................................................................................................
45 3.2.1 p53 shows pulsatile dynamics upon induction of DSBs
.................................................................. 45 3.2.2
p53 response is controlled by three PI3K-like kinases
.................................................................... 47 3.2.3
ATM inhibition has minor effects on p53 dynamics
........................................................................ 49
3.2.4 ATR inhibition has minor effects on p53 dynamics
......................................................................... 50
3.2.5 DNA-PKcs inhibition induces an amplified p53 response
.............................................................. 51 3.2.6 An
increase in unrepaired DSBs is not responsible for changing p53 initial
response .................. 54 3.2.7 Loss of DNA-PKcs activity modulates the
p53 response through prolonged activation of ATM .... 57 3.2.8 There may be a
difference between absent DNA-PKcs and catalytically inactive DNA-PKcs .......
63 3.2.9 MRE11A, RBBP8 and RAD50 are not involved in ATM hyper-activation
...................................... 66 3.2.10 Modulation of p53 dynamics
leads to altered cell fate decision upon DSB induction ................ 67 4
DISCUSSION
.............................................................................................................................................
70 4.1 P53 PROMOTES ENERGY PRODUCTION THROUGH MITOCHONDRIAL RESPIRATION UPON
DNA DAMAGE 70 4.1.1 A systematic study of the role of metabolism in the
response to genotoxic stress ........................... 71 4.1.2 Future
systematic approaches could be done in a breast cancer cell line
...................................... 71 4.2 KALLIKREINS ARE UPREGULATED UPON
GENOTOXIC STRESS IN A P53-DEPENDENT MANNER ............... 72 4.3 HYPER-
ACTIVATION OF ATM UPON DNA-PKCS INHIBITION MODULATES P53 DYNAMICS AND CELL
FATE IN RESPONSE TO DNA DAMAGE
................................................................................................................
74 4.3.1 ATM activity is compensated by the remaining kinases
.................................................................. 74 4.3.2
ATR inhibition has minor effects on p53 dynamics
......................................................................... 74
4.3.3 DNA-PKcs inhibition amplifies the p53 response
........................................................................... 75
4.3.4 Loss of DNA-PKcs activity, but not loss of DNA-PKcs protein, amplifies
p53 response through ATM hyper-
activation...................................................................................................................................
75 4.3.5 The amplified p53 response in cells treated with DNA-PKi leads to
increased cell senescence upon DSB induction
..............................................................................................................................................
77 4.4 CONCLUSIONS
......................................................................................................................................
79 5 SUMMARY – ZUSAMMENFASSUNG
..................................................................................................
80 5.1 SUMMARY
............................................................................................................................................
80 5.2 ZUSAMMENFASSUNG
............................................................................................................................
81 6 BIBLIOGRAPHY
......................................................................................................................................
83 6.1 REFERENCES CONSULTED FOR PLOTTING THE DIFFERENT METABOLIC PATHWAYS AND
MAPPING THE CORRESPONDING GENES AND PROTEINS
.............................................................................................................
97 7 LIST OF PUBLICATIONS
.....................................................................................................................
100 8 APPENDIX
...............................................................................................................................................
101 8.1 LIST OF ABBREVIATIONS
....................................................................................................................
101 8.2 LIST OF CLUSTERS OBTAINED AFTER GENE ENRICHMENT ANALYSIS FOR
BIOLOGICAL PROCESS WITH DAVID ACROSS TIME POINTS
..........................................................................................................................
105 8.3 LIST OF CLUSTERS OBTAINED AFTER GENE ENRICHMENT ANALYSIS FOR
BIOLOGICAL PROCESS WITH DAVID 24 HOURS AFTER IRRADIATION
..........................................................................................................
112
dc.description.abstract
p53 has been called “the guardian of the genome” or “cellular gatekeeper”
because of its prominent role in responding to a wide range of cellular stress
signals. It is a key tumor suppressor in mammalian cells and one of the most
studied human genes, as loss of p53 function is a common feature in the
majority of cancers. Upon genotoxic stress, p53 accumulates in the nucleus and
acts as a transcription factor activating the expression of genes involved in
cell cycle arrest, apoptosis and senescence in order to maintain genome
integrity. Three kinases belonging to the PI3K-like kinase family, ATM, ATR
and DNA-PKcs, are responsible for p53 phosphorylation and stabilization.
However, it is at present unclear how these kinases act in concert to regulate
p53. Using specific inhibitors and quantitative analysis at the single cell
level, the contribution of each kinase for regulating p53 activity upon
genotoxic stress was characterized. ATM and ATR loss was compensated by the
remaining kinases, while DNA-PKcs inhibition led to an amplification in the
p53 response. DNA-PKcs loss caused ATM hyper-activation, which was responsible
for the increased p53 accumulation that sensitized cells for damage-induced
senescence. Although the role of p53 as a tumor suppressor has been mainly
attributed to its ability to induce terminal cell fates, such as senescence,
in recent years p53 has emerged as a regulator of several aspects of cellular
homeostasis including cell metabolism. Many cancer cells primarily use
glycolysis for energy production instead of oxidative phosphorylation (Warburg
effect). Interestingly, p53 has been reported to inhibit glycolysis and
promote mitochondrial respiration through different mechanisms, facts that can
help cells to curb the acquisition of the Warburg effect. However, the way p53
exerts its homeostatic functions is not completely known. In this thesis, a
combination of genome- and proteome-wide approaches were used to
systematically investigate how p53 controls metabolism upon double strand
breaks induction. Several novel genes and proteins controlled by p53 and
involved in pathways related to oxidative phosphorylation were discovered,
especially from fatty acid β-oxidation. Moreover, in this study p53 was
described for the first time as a positive regulator of kallikreins, serine
proteases whose dysregulation is clearly associated with cancer.
de
dc.description.abstract
p53 wurde wegen seiner wichtigen Rolle, auf eine Vielzahl von zellulären
Stress-Signalen zu reagieren, „der Wächter des Genoms” oder „zellulärer
Pförtner” genannt. p53 ist ein Haupttumorsuppressor in Säugerzellen und eines
der am meisten untersuchten menschlichen Gene, da der Verlust der p53-Funktion
ein gemeinsames Merkmal bei den meisten Krebsarten ist. In der zellulären
Signalantwort auf genotoxischen Stress akkumuliert p53 im Zellkern und
reguliert die Expression von Zielgenen, die an Zellzyklusarrest, Apoptose und
Senesenz beteiligt sind, um die Integrität des Genoms aufrechtzuerhalten. Drei
Kinasen, die zur Familie der PI3K-like-Kinasen gehören (ATM, ATR und DNA-
PKcs), sind verantwortlich für die Phosphorylierung und dadurch bedingte
Stabilisierung von p53. Allerdings ist es derzeit unklar, wie diese Kinasen
gemeinsam agieren, um p53 zu regulieren. Unter Verwendung spezifischer
Inhibitoren und quantitativer Analyse auf Einzelzellebene wurde der Effekt
jeder einzelnen Kinase zur Regulierung der p53-Aktivität in der Signalantwort
nach genotoxischem Stress charakterisiert. Der Verlust von ATM und ATR wurde
durch die verbleibenden Kinasen kompensiert, während DNA-PKcs-Hemmung zu einer
Verstärkung in der p53-Antwort geführt hat. Der Verlust von DNA-PKcs
verursacht eine Hyperaktivierung von ATM, die für die erhöhte p53 Akkumulation
verantwortlich ist und Zellen für schadensinduzierte Seneszenz sensibilisiert.
Obwohl die Rolle von p53 als Tumorsuppressor hauptsächlich seiner Fähigkeit
zur Induktion von terminalen Zellschicksalsentscheidungen zugeschrieben wird
(wie Seneszenz), hat sich in den letzten Jahren p53 als Regulator in mehreren
Aspekten der zellulären Homöostase einschließlich des Zellstoffwechsels
herausgestellt. Viele Krebszellen verwenden statt der oxidativen
Phosphorylierung (Warburg-Effekt) in erster Linie Glykolyse zur
Energieerzeugung. Interessanterweise wurde berichtet, p53 würde Glykolyse
hemmen und die mitochondriale Atmung durch verschiedene Mechanismen fördern.
Diese Faktoren können den Warburg-Effekt auf zellulärer Ebene eindämmen.
Jedoch ist die Art und Weise, wie p53 seine homöostatische Funktionen ausübt,
nicht vollständig bekannt. In dieser Arbeit wurde durch eine Kombination von
genom- und proteomweiten Messungen systematisch untersucht, wie p53 nach
Induktion von DNA-Doppelstrangbrüchen den intrazellulären Stoffwechsel
reguliert. Mehrere neue Gene und Proteine, die von p53 kontrolliert werden,
wurden entdeckt. Diese stehen im Zusammenhang mit Signalkaskaden, die an der
oxidativen Phosphorylierung beteiligt sind, insbesondere an der β-Oxidation
von Fettsäure. Außerdem wurde p53 in dieser Arbeit zum ersten Mal als
positiver Regulator von Kallikreins beschrieben, das sind Serinproteasen,
deren Dysregulation eindeutig mit Krebs assoziiert ist.
de
dc.format.extent
115 Seiten
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
genotoxic stress
dc.subject
PI3K-like kinases
dc.subject
time-lapse microscopy
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::572 Biochemie
dc.title
Upstream control and downstream responses of p53 are involved in its tumor
suppression functions upon genotoxic stress
dc.contributor.contact
anna_finz@hotmail.com
dc.contributor.firstReferee
Prof. Dr. Alexander Löwer
dc.contributor.furtherReferee
Prof. Dr. Petra Knaus
dc.date.accepted
2016-12-13
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000103737-4
dc.title.translated
Die Kontrolle der Aktivierung und die Regulation der Zielgene sind an der
Funktion von p53 als Tumorsuppressor nach genotoxischem Stress beteiligt
en
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000103737
refubium.mycore.derivateId
FUDISS_derivate_000000020647
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access