Dottorato di Ricerca in Biologia Cellulare e dello Sviluppo
SAPIENZA
Università di Roma
Facoltà di Scienze Matematiche Fisiche e Naturali
DOTTORATO DI RICERCA
IN BIOLOGIA CELLULARE E DELLO SVILUPPO
XXIV Ciclo
“Yeast as a model for the study of human
p53”
Dottorando
Cristina Piloto
Docente guida
Prof. /Dr Cristina Mazzoni
Tutore
Prof./Dr Loretta Tuosto
Coordinatore
Prof. Rodolfo Negri
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Cristina Piloto
CONTENTS
1.
INTRODUCTION pag 5
1.1.
S. cerevisiae AS A MODEL ORGANISM pag 5
1.2
APOPTOSIS AND AGING pag 10
1.2.1
THE CASPASE YCA1 pag 14
1.2.2 THE ORTHOLOGUE OF THE GENE AIF IN YEAST, aif1
pag 18
1.2.3
THE SERIN PROTEASE Nma111p pag 19
1.2.4
THE YEAST HOMOLOGOUS OF EndoG, Nuc1p pag 21
1.3
THE PHOSPHOPROTEIN p53 pag 24
1.3.1
STRUCTURE OF p53 pag 24
1.3.2
ROLE OF p53 pag 27
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1.3.3
REGULATION OF FUNCTIONS OF p53 pag 39
1.3.4 LOCALIZATION AND REGULATION OF
LOCALIZATION OF p53
pag 51
1.3.5 MUTATIONS OF p53
pag 57
AIM OF THE THESIS
pag 62
2. RESULTS AND DISCUSSION
pag 64
2.1 EXPRESSION OF p53wt AND p53K351N IN YEAST
CELLS
pag 40
2.2 p53wt AND p53K351N INHIBIT THE GROWTH OF
YEAST CELLS
pag 68
2.3 p53wt AND p53K351N INDUCE CELL DEATH WHEN
EXPRESSED IN YEAST CELLS
pag 77
2.4 EFFECTS p53wt AND p53K351N ON YEAST
CHRONOLOGICAL LIFE SPAN
pag 82
2.5 STUDY OF MITOCHONDRIAL MORPHOLOGY UPON
p53 AND p53K351N EXPRESSION
pag 90
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3. CONCLUSIONS
pag 93
4. MATHERIALS AND METHODS
pag 97
5. BIBLIOGRAPHY
pag 102
RINGRAZIAMENTI
pag 141
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1. INTRODUCTION
1.1 S. cerevisiae AS A MODEL ORGANISM
The unicellular yeast
S. cerevisiae is one of the most
intensively used model organism for studying the principles of
microbiology,
characterizing
biochemical
pathways,
and
understanding the biology of more complex eukaryotic organisms.
In fact, many pathways are conserved from yeast to humans,
including DNA replication, recombination, and repair; RNA
transcription and translation; intracellular trafficking; enzymatic
activities of general metabolism, mitochondrial biogenesis,
apoptosis and aging. The reasons that make yeast particularly
suitable for these studies include its easy genetic manipulation,
short generation time, the ability to be maintained in a haploid or
diploid state, ease of mutant isolation, a well-defined genetic
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system, and a highly versatile DNA transformation system that
allow for either the addition of new genes or their deletion.
For all these reasons S. cerevisiae has developed as a very
useful model organism. In fact, many proteins important in human
biology were first discovered by studying their homologs in yeast.
Growth in yeast is synchronized with the growth of the bud,
which reaches the size of the mature cell by the time it separates
from the parent cell. In rapidly growing yeast cultures, all the cells
can be seen to have buds, since bud formation occupies the whole
cell cycle. Both mother and daughter cells can initiate bud
formation before cell separation has occurred. In yeast cultures
growing more slowly, cells lacking buds can be seen, and bud
formation only occupies a part of the cell cycle. The cell cycle in
yeast normally consists of the following stages – G1, S, G2, and M
– which are the normal stages of mitosis.
S. cerevisiae was the first eukaryotic genome that was completely
sequenced. The genome sequence was released in the public
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domain on April 24, 1996. Since then, regular updates have been
maintained at the Saccharomyces Genome Database (SGD). This
database is a highly annotated and cross - referenced databases for
yeast researchers. Another important S. cerevisiae database is
maintained by the Munich Information Center for Protein
Sequences (MIPS). The genome is composed of about 12,156,677
base pairs and 6,275 genes, compactly organized on 16
chromosomes. Only about 5,800 of these are believed to be true
functional genes. Yeast is estimated to share about 23% of its
genome with that of humans (Williams N, 1996).
The availability of the S. cerevisiae genome sequence and
the complete set of deletion mutants has further enhanced the
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power of S. cerevisiae as a model for understanding the regulation
of eukaryotic cells.
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Figure 1.1.1 Picture showing the structure of budding yeast
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1.2 APOPTOSIS AND AGING
Apoptosis is a highly regulated cellular suicide program
crucial for metazoan. However, disfunction of apoptosis also leads
to several diseases (Madeo et al., 2004). Indeed, misregulation of
apoptosis can result in human diseases such as neurodegenerative
disorders, spread of viral infections, AIDS and cancer (Herker et
al., 2004). During the past years, yeast has been successfully
established as a model to study mechanisms of apoptosis regulation
(Herker et al., 2004).
Typical markers of apoptosis are DNA fragmentation,
phosphatidylserine externalization and chromatin condensation.
These markers, as shown in figure 1.2.2, are conserved in yeast.
Further, a caspase - like protease (Yca1p) mediates oxygenstress-dependent apoptosis in yeast.
In mammals, there are two pathways of apoptosis that
leading to caspase-dependent apoptosis: the intrinsic apoptotic
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pathway and the extrinsic apoptotic pathway. The intrinsic
pathway is characterized by mitochondrial outer membrane
permeabilization (MOMP) that is a crucial step in the intrinsic
apoptotic pathway in mammalian cells (Mazzoni and Falcone,
2008), while the extrinsic pathway begins outside the cell through
the activation of specific pro-apoptotic receptors on the cell
surface. These are activated by specific molecules known as proapoptotic ligands. These ligands include Apo2L/TRAIL and
CD95L/FasL and bind their cognate receptors DR4/DR5/Fas,
respectively. Unlike the intrinsic pathway, the extrinsic pathway
triggers apoptosis independently of the p53 protein.
Two complementary and partly overlapping models of
aging have been investigated in yeast: the replicative life span
model and the chronological life span model.
Chronological life span is defined as the length of time
cells survive after they are driven into a non-dividing state by
depletion of nutrients from the medium, while replicative life span
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is determined by counting the number of times cells divide in the
presence of nutrients before they senescence and die.
Both chronologically and replicatively aged yeast cells dye
exhibit typical markers of apoptosis, accumulate oxygen radicals,
and show caspase activation (Herker et al., 2004).
Apoptosis in yeast confers a selective advantage for this
unicellular organism, and it has been demonstrated that old yeast
cells release substances into the medium that stimulate survival of
the clone (altruistic suicide) (Herker et al., 2004)..
In recent years, several homologues of classical apoptotic
regulators have been identified and characterized, i.e. caspase
(YCA1) (REF), AIF1 (REF), EndoG (endonuclease G, NUC1)
(REF) and
Omi (NMA111)(REF), suggesting that the basic
machinery of apoptosis is indeed present and functional also in
yeast.
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Figure 1.2.1 DAPI staining shows nuclear fragmentation, TUNEL assay shows
fragmentation of DNA, the exposition of the phosphatydilserine was observed
through annexin V and electron micrographs show condensation of chromatin.
This picture shows the importance of the conserved features between yeast and
mammalian. n nucleus, m mitochondria v vacuole.
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1.2.1 THE CASPASE YCA1
Caspases are members of a family, or structurally related
group, known as cysteine proteases. The name caspase derives
from cysteine-dependent aspartate specific protease: catalysis is
driven by a critical conserved cysteine side chain of the enzyme,
and by a stringent specificity for cleaving protein substrates
containing aspartic acid. The activation of caspases has been
recognized as one of the key processes linked to apoptosis in
mammalian cells in which several caspases, grouped into two
classes, have been described (Mazzoni and Falcone, 2008).
The analysis of the S. cerevisiae genome did not reveal the
presence of caspases, but by sequence comparison ORF YOR197
was identified coding for a caspase-like protein, fitting into the
type I category of metacaspases, and it was named MCA1 (Uren et
al, 2000; Szallies et al., 2002).
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Following
studies
demonstrated
that,
following
an
apoptotic stimulus, the product of MCA1 was processed by the
proteolytic removal of a 14 kDa peptide leading, as in mammalian
caspases, to the activation of the metacaspase.
Moreover, in the same study, it has been demonstrated that
the conserved Cys297 residue is necessary for the proteolytic
cleavage and is important for the fully activity of the caspase
(Madeo et al., 2002).
Lysates from yeast over - expressing the MCA1 gene could
efficiently cleave IETD-AMC peptide, a caspase substrates typical
of initiator caspases (i.e. caspase 8), while no activity was found
toward DEVDAMC, a characteristic substrate for effector caspases
(i.e. caspase 3) suggesting that MCA1 could operate as the initiator
caspase 8. The over - expression of MCA1 rendered cells more
susceptible to cell death following an apoptotic stimulus, being cell
death completely abolished in the presence of the caspase inhibitor
zVAD-fmk. Conversely, yeast mutants lacking the MCA1 gene
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survived to hydrogen peroxide treatment and to aging better than
wild type cells (Madeo et al., 2002).
From these results, Yor197w was proposed as a bona fide
caspase gene in S. cerevisiae and it was renamed YCA1 (Yeast
CAspase) (Madeo et al., 2002).
The apoptotic events observed in S. cerevisiae, due to
external stimuli as well as internal signals, were often dependent
on the activity of the yeast metacaspase Yca1p while, in other
cases, cell death occurred also in the absence of this activity.
Disruption of the apoptotic machinery by deletion of YCA1
increases the survival in long term cultures but it is
disadvantageous for the population as, in direct competition assays,
the wild type cells outlast the disruptants (Herker et al., 2004). An
aged yca1 - null mutant strain is no longer able to regrow when
nutrients become available after a period of starvation, leading to a
population with a high percentage of damaged and old cells. This
indicates a physiological role of apoptosis in yeast essential to
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operate a selection for the fitter cells in aging - related cell death
(Buttner et al., 2006).
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1.2.2 THE ORTHOLOGUE OF THE GENE AIF IN
YEAST, aif1
Apoptosis - inducing factor (AIF) in mammalian cells is a
flavoprotein with oxido-reductase activity localized in the
mithochondrial intermembrane space (Susin et al., 1999; Miramar
et al., 2001). Upon apoptosis induction, AIF translocates to the
nucleus, where it leads to chromatin condensation and DNA
degradation. AIF has been suggested to control caspaseindependent
pathway
of
apoptosis,
important
for
neurodegeneration and normal development (Susin et al., 1999;
Cregan et al., 2002).
An orthologue of AIF has been described in yeast cells.
Yeast Aif1p shows the same localization and exhibits similar death
executing pathways as mammalian AIF. Further Aif1p is
dependent on cyclophilin A (CypA) and partially on caspase action
(Wissing et al., 2004).
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1.2.3 THE SERIN PROTEASE Nma 111p (Omi)
In the yeast Saccharomyces cerevisiae Nma111p functions
as a nuclear serine protease that is necessary for apoptosis under
cellular stress conditions, such as elevate temperature or treatment
of cells with hydrogen peroxide to induce cell death.
The role of nuclear protein import in the function of
Nma111p in apoptosis has been examined.
Nma111p contains two small clusters of basic residues
towards its N-terminus, both of which are necessary for efficient
translocation into the nucleus. Nma111p does not shuttle between
the nucleus and cytoplasm during either normal growth conditions
or under environmental stresses that induce apoptosis.
The N-terminal half of Nma111p is sufficient to provide the
apoptosis - inducing activity of the protein, and the nuclear
localization signal (NLS) sequences and catalytic serine 235 are
both necessary for this function. Now there is evidence that
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intranuclear Nma111p activity is necessary for apoptosis in yeast
(Belanger et al., 2009).
Nma111p is a nuclear protein that interacts with nuclear pore complexes (NPCs) (Fahrenkrog et al., 2004). Both Yca1p, the
yeast caspase, and Bir1p, the only identified substrate for
Nma111p, are also nuclear proteins (Walter et al., 2006).
Therefore, some of the processes that occur in the cytoplam during
mammalian apoptosis seem to occur in the nucleus during yeast
cell death. The importance of the nucleus for the yeast apoptotic
programme is further supported by the notion that Aif1p and
Nuc1p move from mitochondria to the nucleus upon induction of
apoptosis (Buttner et al., 2007; Wissing et al., 2004).
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1.2.4 THE YEAST HOMOLOGOUS OF EndoG,
Nuc1p
Endonuclease G (EndoG) is located in mitochondria yet
translocates into the nucleus of apoptotic cells during human
degenerative diseases. Nonetheless, a direct involvement of
EndoG in cell - death execution remains equivocal, and the
mechanism for mitochondrio-nuclear translocation is not known.
Has been shown that the yeast homologous of EndoG
(Nuc1p) can efficiently trigger apoptotic cell death when excluded
from
mitochondria.
Nuc1p
induces
apoptosis
in
yeast
independently of metacaspase or of apoptosis inducing factor
AIF1. Instead, the permeability transition pore, karyopherin
Kap123p, and histone H2B interact with Nuc1p and are required
for cell death upon Nuc1p overexpression, suggesting a pathway in
which mitochondrial pore opening, nuclear import, and chromatin
association are successively involved in EndoG - mediated death.
Deletion of NUC1 diminishes apoptotic death when mitochondrial
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respiration is increased but enhances necrotic death when oxidative
phosphorylation is repressed, pointing to dual – lethal and vital –
roles of EndoG (Buttner et al., 2007).
EndoG has been described as a mitochondrial nuclease that
digests both DNA and RNA (Ruiz-Carrillo and Renaud, 1987;
Schafer et al., 2004). Upon apoptosis induction, translocation of
mammalian EndoG to the nucleus coincides with large - scale
DNA fragmentation (Li et al., 2001; Parrish et al., 2001).
Recently, nuclear translocation of EndoG has been associated with
progression of several degenerative disorders, such as cerebral
ischemia (Lee et al., 2005) and muscle atrophy (Leeuwen-burgh et
al., 2005).
Similar to other mitochondrially located cell - death
regulators like cytochrome c or apoptosis inducing factor (AIF)
(Cheng et al., 2006; Vahsen et al., 2004), EndoG may have a
genuine vital function, besides its proapoptotic function. In fact,
EndoG was suggested to play a role in cell proliferation and
replication (Huang et al., 2006)
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Mitochondria
are
highly
dynamic
organelles
that
continuously fuse and divide. In many organisms, including worms
and mammals, mitochondrial division promotes the release of
cytochrome c to trigger apoptosis. Furthermore, mitochondrial
dynamics is thought to counteract aging and constitute an
organellar quality control mechanism.
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1.3 THE TUMOUR SUPPRESSOR p53
1.3.1 STRUCTURE OF p53
The human p53 gene was shown to span about 20 kb of
DNA and to be localized to the short arm of chromosome 17
(17p13). This gene is composed of 11 exons, the first of which is
non coding and localized 9-10 kb away from exons 2 through 11
(Benchimol et al., 1985; Isobe et al., 1986; Oren, 1985).
The p53 has been conserved during evolution (Soussi et al.,
1987). Through cross-species comparison of amino acid sequences,
the p53 proteins showed the existence of five highly conserved
regions within the amino acid residues 13-23, 117-142, 171-181,
234-250 and 270-286 (Soussi et al., 1990; Soussi and May, 1996).
These regions, termed domains I-V, were expected to be crucial for
the p53 functions.
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The human p53 protein is a transcription factor, consists of
393 amino acids and contains four major functional domains: : the
activation domain (AD, residues 1-45), a prolin rich domain, the
DNA binding domain (DBD, residues 100-300) and the
tetramerization domain (TD, 324-356).
Whitin the N-terminus are the transactivation domain and a
proline-rich region, which is required for apoptotic function.
Whitin the N-terminus are the interaction sites of p53 with
components of the transcriptional machinery as well as ubiquitin
ligase Mdm2. In the central domain harboring the sequence
specific DBD domain occur most of the tumor associated
mutations. This central region also contains binding sites for
interaction with members of the Bcl2 protein family. The Cterminal region contains the oligomerization domain as well as
nuclear localization and export signals. Several sites within the Nterminal region have been shown to be phosphorylated and the Cterminal region contains numerous sites of modification which
influence stability, localization and activity of p53.
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Figure 1.3.1 Diagram showing the domain structure of the p53
protein (Yee and Vousden, 2005)
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1.3.2 ROLE OF p53
p53 may be considered the best known of all tumor
suppressors. The loss or inhibition of p53 activity and functions
prevents cellular senescence and apoptosis, thus favoring cancer
development and progression. p53 is indeed mutated in 50%
human cancers.
In normal cells, p53 levels are maintained low by the
activity of the E3 ubiquitin ligase MDM2. Many types of stress
signals, (i.e. DNA damage, oncogene activation, hypoxia-anoxia)
increase the levels of p53 by inducing its transcription and/or by
stabilizing the protein.
p53 is a transcription factor that direcly activates the
expression of both cell cycle and apoptosis regulating genes
(Vousden and Lu, 2002). Over 4,000 putative p53-binding sites
were identified in several gene promoters.. Whether all of these
will prove to be bona fide target genes of p53 is not known.
Although far from complete, these examples illustrate that many of
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the genes that are induced by p53 can be divided into groups that
might mediate a specific p53 function, such as inhibition of cell
growth, DNA repair, activation of apoptosis or regulation of
angiogenesis (Vousden and Lu, 2002).
The tumour suppressor function of p53 depends principally
on its abilty to prevent cellular proliferation in response to stress
stimuli that are encountered during tumourigenic progression.
Activation of p53 leads to cell cycle arrest and apoptosis, and can
play a role in the induction of differentiation and cellular
senescence (Almog and Rotter, 1997; Lundberg et al., 2000). Wild
- type p53 has been shown to inhibit angiogenesis in tumour, by
activating or repressing genes that regulate new blood vessels
formation (Dameron et al., 1994; Nishimori et al., 1997; Bouvet et
al., 1998). p53 can also play a direct role in the repair of DNA
damage, both through nucleotide excision repair and base excision
repair ( Wani et al., 1999; Offer et al., 2001).
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
Function of cell cycle arrest of p53
The cell cycle arrest function of p53 correlates well with its
ability to function as a trancription factor (Crook et al., 1994;
Pietenpol et al., 1994).
Waf/Cip1
genes is p21
One of cell cycle regulating p53 target
Waf/Cip1
(El-Deiry et al., 1993). p21
is a
cyclin-dependent kinase inhibitor that can activate both G1 and G2
cell cycle arrest similar to those seen in response to p53 induction (
Agarwal et al., 1995; Bates et al., 1998). Importantly, , p21WAf/Cip1
-deficient cell are defects in both G1 and G2 arrest, DNA synthesis
and mitosis. Another target of p53 that contributes to the p53induced G2 arrest is 14-3-3 σ (Hermeking et al., 1997). The 14-3-3
family proteins play a role in signal transduction and cell cycle
control (Musling and Xing, 2000). Although 14-3-3 σ - deficient
cells may transiently arrest in G2 phase after DNA damage, they
are unable to maintain the cell cycle arrest status. Further potential
mediators of the G2 arrest include GADD45 and Reprimo (Ohki et
al., 2000).
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Recent data implicate p53 as part of a checkpoint that
senses microtubule disruption, and that prevents the outgrowth of
aneuploid variants that emerge after exposure of cells to
microtubule inhibitors.
A key link was established when Brian Reid and colleagues
demonstrated that in rodent cells microtubule disruption in
combination with p53 loss allows downstream events (DNA
replication) to occur prior to the successful completion of upstream
events (mitosis) (Cross et al., 1995). The consequence of this loss
of coordination is an increase in the number of cells with greater
than 4N DNA content. Thus, loss of p53 leads to DNA
reduplication after exposure to agents that inhibit mitotic division,
such as nocodazole and colcemid. These data led to the proposal
that p53 functions in a mitotic spindle checkpoint in which it
monitors and prevents exit from mitosis under conditions that
hinder cell division (Cross et al., 1995).
Furthermore, p53 contributes either to repair or cell death
by stimulating expression of either pro - or antioxidant genes.
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Figure 1.3.2 p53 drives different responses under conditions of low stress
(where cell survival and repair is supported) and high stress (where the
damaged cell is eliminated through death or senescence) (Gottlieb and
Vousden, 2010).
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 p53 pro-apoptotic function
In most cell types, the apoptotic response reflects the
cumulative action of several p53-induced signals. The ability to
activate apoptosis through different apoptotic pathways may be
relevant for the tumour-suppressor activity of p53 (Vousden and
Lu, 2002).
The concept that apoptosis is a cumulative response to
many signals, including p53, indicates that loss of any of these
might enhance survival by reducing the apoptotic burden to below
the threshold that is necessary for the execution of death (Vousden
and Lu, 2002).
Recent studies have suggested that the straightforward role
of p53 as a transcription factor that eliminates developing tumor
cells by inducing the expression of apoptotic target genes is only
part of a much more complicated story. There is now a firm body
of evidence supporting a transcriptionally independent activity that
involve a direct role of p53 in mitochondria, thus favoring the
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release of cytocrome C and the activation of the Apaf-1/caspase-9
apoptotic cascade (Yee and Vousden, 2005).
A number of early studies suggest that p53 may have an
apoptotic function that is completely separate from the regulation
of gene expression. An explanation of this activity has been
provided by the recent observations that, following stress, a
proportion of p53 appears to function outside the nucleus, in the
cytoplasm or at the mitochondria, and that p53 can be found in
association with several members of the Bcl2 family of proteins.
The Bcl2 family proteins are key components of the intrinsic
apoptotic pathway and fall into three main groups; the proapototc
effectors proteins (Bax and Bak), which directly perturb
mitochondrial membrane potential, the BH-3 only proteins that
directly or indirectly drive the activation of Bax and Bak, and the
anti-apoptotic proteins that sequester the pro-apoptotic family
members and hold them inactive (such as Bcl2, BclxL and Mc11).
The BH-3 only proteins are further divided into two groups; the
“activators” like Bim and Bid that directly bind Bax and Bak to
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activate them and the “enablers” like Bad and Bik that bind the
anti-apoptotic
family
members
to
release
the
activators.
Unexpectedly, p53 may function in a manner analogous to the
BH3-only proteins, with evidence to support two broad, but not
mutually exclusive, models. In the first, p53 acts as an “enabler”
BH3 domain protein, interacting with the anti-apoptotic proteins
and presumably releasing pro-apoptotic BH3 domain proteins to
drive apoptosis. The second model is based on the observation that
p53 may function as an “activator” BH3-only proteins, by directly
activating the apoptotic functions of Bax or Bak (Yee and Vousden,
2005). Indeed, p53 protein itself can localize to the mitochondria
presenting a potential additional transcription-independent way of
mediating apoptosis (Marchenko et al., 2000).
Recently, other pro-apoptotic members of the BCL2-family
named NOXA (Oda et al., 2000) and PUMA (Nakano and
Vousden, 2001; Yu et al., 2001) have been identified as p53 targets.
These proteins, as well as another p53 target gene product,
p53AIP1 (Oda et al., 2000), localize to the mitochondria and
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promote loss of the mitochondrial membrane potential and
cytochrome c release (Bossy-Wetzel and Green, 1999).
Perturbation of mitochondrial integrity may also be
mediated by several genes encoding for redox-controlling
enzymes, which were identified as p53-induced genes (PIGs). It
has been proposed that reactive oxygen species (ROS) produced by
these PIGs cause damage to mitochondria, which in turns initiate
apoptosis.
p53 has been also implicated in the regulation of the
extrinsic death receptor-induced pathway of apoptosis. The
expression of at least two death receptors, FAS/FASLand
DR5/KILLER, is up-regulated by p53 (Owen-Schaub et al., 1995;
Wu et al., 1997). The activation of FAS and DR5 death-receptors
by their ligands FASL and TRAIL respectively, results in their
trimerization and recruitment of intracellular adapter molecules
which initiate the caspase-8/10 cascade and apoptosis (Ashkenazi
and Dixit, 1998).The DR5 promoter is a direct target of p53
(Takimoto and El-Deiry, 2000), while cell surface expression of
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FAS is enhanced by p53 that promotes its trafficking from Golgi to
the plasma membrane (Bennett et al., 1998).
The loss of survival signalling can also augment p53mediated apoptosis (Gottlieb et al., 1994; Abrahamson et al., 1995;
Canman et al., 1995; Lin and Benchimol, 1995; Prisco et al., 1997)
and p53 may both negatively regulate the growth factor (i.e insulin
growth factor, IGF) signalling pathway (Buckbinder et al., 1995)
and inhibit integrin-associated survival signalling thus further
sensitizing cells to apoptosis (Bachelder et al., 1999). The NF-κB
transcription factor has lately been shown to play an important role
in p53-mediated - apoptosis (Ryan et al., 2000)
In conclusion, several cellular responses to p53 activation
have been described, and the choice of response depends on factors
such as cell type, cell environment and other oncogenic alterations
that are sustained by the cell. In general, however, the effects of
p53 activation is to inhibit cell growth, either through cell cycle
arrest or induction of apoptosis, thereby preventing tumor
development (Vousden and Lu, 2002).
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Indeed, under some circustances, when the damage is
reversible, p53 contributes to the repair of genotoxic damage,
blocking cell cycle, potentially allowing for the release of the
rehabilitated cell back into proliferating pool. On the other hand,
rather in most cases, when the cell damage is extensive, induction
of p53 leads to an irreversible inhibition of cell growth, most
decisevely by activating apoptosis.
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Figure 1.3.3
p53 induces apoptosis by activating target genes such as
PIGs,Bax, Noxa; PUMA, p53AIP1, FAS PIDD and IGF-BP3. p53-induced
apoptosis involves change in mitochondrial membrane potential (ΔY),
cytochrome C release and caspase activation (Bálint and Vousden, 2001).
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1.3.3 REGULATION OF FUNCTIONS OF p53
Since p53 is a powerful inhibitor of cell proliferation,
control of its activity is essential during normal growth and
development. Regulation of p53 has been described at the level of
transcription, translation, conformational changes, and various
covalent and non-covalent modifications. At present, it is clear that
one of the key mechanisms by which p53 functions are regulated,
relies on the control of protein stability. MDM2 plays a pivotal role
in regulating p53 ubiquitination and degradation and multiple
forms of stress stimuli activate p53 by countering MDM2mediated degradation of p53 (Ryan et al., 2000).
Two related proteins – MDM2 and MDMX (MDM4) –
have been described as crucial in regulating p53 levels. MDM2,
which participates in an autoregulatory loop with p53, contains a
RING finger domain and, similarly to many RING finger proteins
has been shown to function as a E3 - ubiquitin ligase that targets
p53 for degradation. MDM2 targets both itself and p53 for
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Cristina Piloto
ubiquitination, and mutations in its RING finger domain lead to the
stabilization of both proteins (Honda et al., 1997). The activation
and stabilization of p53 is generally associated with inhibition of
the function of MDM2, and defects in the pathways that curb
MDM2 activity are common in tumours that retain wild-type p53
(Vousden and Lu, 2002). Interestingly, the transcriptional coactivator p300/CBP that functions in acetylating and activating
p53, also participates in the degradation of p53 by MDM2,
possibly by acting as a platform to allow efficient p53/MDM2
interaction.
MDM2 has been shown to inhibit p53 activity in several
ways: by binding to the transactivation domain of p53, by targeting
p53 for ubiquitination, by inhibiting acetylation of p53 and by
shuttling p53 to cytoplasm. The importance of MDM2 in the
regulation of p53 activity is illustrated by the observation that
MDM2 deficiency causes early embryonic lethality in mice, an
event that may be rescued in a p53 - null background. Conversely,
amplification of MDM2 is associated with the development of
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tumours that retain wild - type p53 suggesting that overexpression
of MDM2 prevents the normal p53 - mediated response to
oncogenic stress (Ryan et al., 2000).
 Mechanisms regulating p53 function by MDM2
Binding of MDM2 to p53 and its
ubiquitin-dependent degradation
MDM2 interacts with the N-terminal region of p53, which
also contains the major acidic transcriptional activation domain
(Momand et al., 1992). This binding of MDM2 to p53 can inhibit
p53's transcriptional activity by impairing the association of p53
with transcriptional coactivators such as p300/CBP.
The binding of MDM2 with p53 plays an additional
important role in controlling p53 function by contributing to the
regulation of p53 protein stability (Kubbutat et al., 1997). In most
unstressed tissues p53 is expressed at very low levels are
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maintained in large part by the costant removal of newly
sinthesized p53 through ubiquitin-dependent degradation by the
proteasome. Ubiquitin - modification of proteins involves a
multienzyme cascade, with substrate specificity determined by a
large group of ubiquitin protein ligase (E3). One recently described
group of E3 ligases are RING-finger proteins, which are thought to
serve
as a bridge to allow transfer of ubiquitin from the E2
ubiquitin-conjugating enzyme to the substrate (Joazeiro and
Weissman, 2000). MDM2 is a RING-finger protein that functions
as a ubiquitin ligase for both p53 and itself (Honda et al., 1997;
Midgley et al., 2000).
Mutations within the RING-finger domain of MDM2
inhibit the ubiquitination of p53 and MDM2 itself resulting in the
stabilization of both proteins. Interestingly, the ability of MDM2 to
target p53 for degradation is also regulated by other factors. A role
for p300/CBP has been described in supporting efficient
degradation of p53, probably by facilitating the interaction between
p53 and MDM2 in cells, although this activity depends on an
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interaction between these proteins different from those involving
the p53 transactivation domain.
Nuclear export of p53 by MDM2

Although ubiquitination of p53 by MDM2 has the main
function to target the p53 protein to the proteasome, this activity
may also allow the export of p53 from the nucleus to the
cytoplasm, where degradation of p53 takes place.
 Other mechanisms regulating p53 function
Modifications of p53

•
Phosphorylation
Phosphorylation sites in p53 are clustered within the N- and
C-termini and include potential targets for numerous cellular
kinases (Meek, 1998). Phosphorylation within to the N-terminal
MDM2 binding domain in p53 is mostly involved in the regulation
of p53 stability, by interfering with the ability to bind to MDM2.
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Several stress-response kinases such as ATM, ATR, Chk1, and
Chk2 have been shown to phosphorylate this region of p53, and
inhibition or loss of these kinases renders cells deficient in
mounting a p53 response to stress signals. Both ATM and ATR
appear to have a multiples roles in allowing activation of p53 in
response to DNA damage. These kinases directly phosphorylate
p53 on serine 15 and 37, thereby enhancing the transcriptional
activity of p53 ( Sakaguchi et al., 1998; Dumaz and Meek, 1999),
ATM also activate Chk2, a kinase that phosphorylate p53 directly
within the MDM2 binding domain, thus reducing binding to
MDM2 (Hirao et al., 2000). ATM activation also induces
dephosphorylation of the C-terminus of p53, and ATM can also
directly phosphorylate MDM2 (Khosravi et al., 1999), although the
consequences of this activity are less understood. Many other
kinases have been shown to be capable of phosphorylating the Nterminus of p53, including DNA-PK, JNK, p38, CK1 and CAK
(Ljungman, 2000).
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Many proteins that shuttle between the cytoplasm
and nucleus are regulated by phosphorylation mediated by CK or
Cdc2/cyclin B at sites near their NLS (Nuclear Localization
Signal) and NES ((Nuclear Export Signal) domains (Vandromme et
al., 1996). While phosphorylation of these proteins by CK is
associated
with
nuclear
localization,
phosphorylation
by
Cdc2/cyclin B is associated with cytoplasmic localization. The C
terminal domain of p53 has both Cdc2/cyclin B and CK
phosphorylation sites. Phosphorylation of ser392 by CK has been
shown to stimulate tetramerization of p53 (Sakaguchi et al., 1997).
Since tetramerization of p53 has been suggested to hide the NES of
p53 and thus block nuclear export (Stommel et al., 1999)),
phosphorylation of p53 by CK may stimulate nuclear accumulation
of p53 by blocking p53’s export. In contrast, phosphorylation
induced by Cdc2/cyclin B is associated with cytoplasmic
localization of many different types of proteins. It has been shown
that
the
tetramerization
stimulating
effect
of
ser392
phosphorylation by CK II can be blocked by phosphorylation of
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ser315 by Cdc2/cyclin B (Sakaguchi et al., 1997). Thus, it is
possible that the nucleocytoplasmic shuttling of p53 could be
regulated, in part, by these two kinases where CK may stimulate
nuclear import and Cdc2/cyclin B may stimulate nuclear export
(Ljungman, 2000).
•
Acetylation
p300/CBP has been shown to enhance p53's transcriptional
activity by interacting with the N-terminus of p53 and acetylating
lysine 382 in the C-terminus of the p53 protein (Sakaguchi et al.,
1998). The acetylation of this site activates p53's binding function,
thus reinforcing the role of p300/CBP as a key coactivator for p53
transcritptional activity. Interestingly, phosphorylation of p53
within the N-terminus by kinase like ATM or ATR enhances
p300/CBP binding and C-terminal acetylation, revealing a complex
pathway in each multiple codependent modifications of p53 may
be necessary for full activation. The inhibition of acetylation by
either histone deacetylases (Fogal et al., 2000) or MDM2 (Kobet et
al., 2000) results in a reduction in p53's transcriptional activity.
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•
Sumoylation and other modifications
of p53
SUMO is a small ubiquitin-related
protein that is
conjugated to target proteins through enzyme cascades similar to
those responsible for ubiquitination. p53 is modified by SUMO at
one of the lysine residues that is also ubiquitinated, and although
SUMO modification does not regulate p53 stability, this
modification enhances p53's transcriptional activity (Gostissa et
al., 1999). Interestingly this modification of p53 may also be
regulated by phosphorylation, again illustrating the complex
codependence of regulatory modifications of p53. Several other
modifications of p53 have been described to affect both both
protein stability and functions, including glycosilation and
ribosylation by PARP (Vaziri et al., 1997; Kumari et al., 1998),
which might affect both protein stability and protein function.
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Cristina Piloto
Interaction of p53 with other proteins
•
Many p53-interacting proteins that might play a role in the
stabilization and activation of p53 have been described (Prives et
al., 1999). Interaction of p53 with Ref-1 (Jayaraman et al., 1997)
and HMG-1 (Jayaraman et al., 1998) leads to the activation of
p53, possibly whithout requirement for covalent modification of
the p53 protein. Numerous other proteins such as WT1, BRCA1,
WRN, E2F1, and pRB, have been shown to augment p53 function,
either by stabilizing the p53 proteins or by activating p53's
transcriptional activity (Sionov and Haupt, 1999).
The ARF protein
•
Another mechanism to stabilize p53 in response to stress
involves the protein ARF. The alternative reading frame product of
ARF
the INK4A locus, called p14
ARF
(mouse p19
) binds directly
to MDM2, inhibiting p53 ubiqutination by MDM2 (Bates et al.,
1998; Kamijo et al., 1998). In some systems, ARF also leads to the
relocalization of MDM2 from the nucleoplasm to the nucleolus,
through nucleolar localization signals present in both ARF and
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MDM2 (Lohrum et al., 2001). As a consequence, p53 is efficiently
stabilized and activated.
The importance of the ARF/p53 pathways is illustrated in
mice, where deletion of ARF results in tumour development
(Kamijo et al., 1997). Although mutations of ARF have been rarely
found in human tumours, the loss of ARF expression by either
methylation of the ARF promoter (Esteller, 2000)
•
The role of MDMX
Another regulator of p53 stability is the protein MDMX.
MDMX, a protein related to MDM2, also possesses a p53-binding
domain and a RING-finger domain. Binding of MDMX inhibits
p53 transactivation domain. Binding of MDMX inhibits p53
transactivation function (Shvarts et al., 1996) through a mechanism
independent of p53 for degradation. It is possible that MDMX does
not show ubiquitin ligase activity, and hetero-oligomerization of
MDMX with MDM2 through their RING finger domain results in
the stabilization of MDM2 (Sharp et al., 1999; Tanimura et al.,
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1999) but protects p53 by MDM2-mediated ubiquitination and
degradation (Jackson and Berberich, 2000).
Figure 1.3.4 Cartoon depicting the p53 protein, showing the major
functional domains, sites of interactions with some other proteins and
regions of covalent modifications (Woods and Vousden, 2001).
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1.3.4 LOCALIZATION AND REGULATION OF
LOCALIZATION OF p53
In the past few years it has become clear that in addition to
modulation of protein stability, p53 function is also regulated by its
subcellular localization. Since one of the key functions of p53 is
the regulation of transcription, localization of p53 in the nucleus
plays an important role for the p53 response. Active transport of
p53 towards the nucleus by dynein and microtubule network has
been described, and several nuclear localization signals in the Cterminus of p53 can contribute to nuclear import. Once enetered
into the nucleus, regulatory mechanisms exist to control the export
of p53 back out to the cytoplasm.
p53 nuclear accumulation in resposne to certain stress
stimuli, such as γ-irradiation, is cell cycle-dependent (Komarova et
al., 1997). On the contrary, other stresses, such as hydrogen
peroxide and heat shock, induce p53 nuclear translocation and
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Cristina Piloto
accumulation independently of cell cycle phase (Sugano et al.,
1995).
The microtubule network shows an inherent polarity from
the dynamic “plus” end (usually at the cell periphery) to the
relatively stable “minus” end (at the centrosome, usually near, or
even linked to, the nucleus) (Vousden and Woude, 2000).
The study of Giannakakou and colleagues shows that
transport of p53 along the microtubules towards the nucleus occurs
only in response to stress, and that disruption of the microtubule
network or interference with dynein function impairs localization
of p53 to the nucleus, thus inhibiting p53 activation ( Vousden and
Woude, 2000).
Although, the accumulation of p53 into getting p53 into the
nucleus is obviously important in allowing p53 to function,
dampening p53 activity in normally dividing cells seems to require
transport out of the nucleus (Vousden and Woude, 2000) .
Besides direct ubiquitination of p53, MDM2 also plays a
role in regulating the subcellular localization of p53, driving
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degradation of p53 through the proteasome. Several lines of
evidence indicate that this degradation of p53 occurs in the
cytoplasm, although both MDM2 and p53 are nuclear proteins
(Vousden and Woude, 2000) . MDM2's ubiquitin ligase activity
contributes to the efficient nuclear export of p53 (Boyd et al., 2000;
Geyer et al., 2000), which depends on the nuclear export sequence
(NES) identified in the C-terminus of p53 (Stommel et al., 1999). It
is possible that the ubiquitination of p53 reveals the NES, possibly
by driving p53 into a monomeric form (Stommel et al., 1999),
allowing access to the nuclear export machinery (Bálint and
Vousden, 2001).
Several models of how p53 is exported from the nucleus
have been put forward. MDM2 contains nuclear-export signals in
p53 has complicated this model, and there is some evidence that
the export of p53 continues unabated even in the absence of
MDM2. Now a further wrinkle on the system has been uncovered
by two studies that have shown that the ubiquitin-ligase activity of
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Cristina Piloto
MDM2 is necessary for p53 export, but export of MDM2 itself is
not required (Boyd et al., 2000; Geyer et al., 2000).
Together, the studies indicate that, in the absence of stress
signals, p53 activity may be regulated both by suppressing
localization to the nucleus and by enhancing transport out of the
nucleus. In response to the appropriate signals, nuclear import of
p53 is activated, whereas export is inhibited, so maximing the
accumulation of p53 in the nucleus. In tumours that retain wildtype p53 (roughly 50%), defects in either of these pathways
impinge on the ability to activate the p53 response, several tumourassociated defects have been described that lead to a failure to
block MDM2 function in response to stress signals, and so result in
an inability to stop the nuclear export and degradation of p53
(Vousden and Woude, 2000).
Although previous studies have shown that cytoplasmic
localization of p53 in tumour cells may be the result of a
hyperactive nuclear-export system, the study by Giannakakou and
colleagues raises the possibility that cytoplasmic retention may
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also result from defects in the ability of p53 to associate with
microtubules in response to stress (Vousden and Woude, 2000).
The importance of microtubules in p53 function may have
important repercussions in tumour therapy, as microtubuledisrupting drugs like taxol are commonly used for chemotherapy
(Vousden and Woude, 2000).
Figure 1.3.5 Domain structure of p53 and MDM2 proteins. NLS, nuclear
localization signal; NES, nuclear export signal; NoLs, nucleolar localization
signal (Bálint and Vousden, 2001).
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Cristina Piloto
Figura 1.3.6 Model for regulation of the subcellular localization of
p53. Association with microtubules directs p53 towards the nucleus, using
the minus-end-directed molecular motor dynein, where import receptors
recognize localization signals in the C-terminus of p53. Export from the
nucleus requires nuclear-export sequences in p53 and MDM2 activity,
possibly through ubiquitination of p53. Defects in the import pathway, or
failure to inhibit the export pathway, could compromise the ability to
activate p53 and contribute to tumour development. Ub, ubiquitin. (Vousden
and Woude, 2000).
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1.3.5 MUTATIONS OF p53
From the data available, it would seem that only 5% of
TP53 mutations are found in the regulatory domains (amino
terminus, amino acids 1-99; carboxyl terminus, aminoacids 301393), whereas 95% of the mutations occur in the central region of
TP53, which is responsible for sequence-specific DNA binding
(aminoacids 100-300) (Vousden and Lu, 2002).
Moreover, mutations within the TD (tetramerization
domain) have been recently reported and associated with human
tumors. The TD of p53 is essential for efficient p53 trancriptional
activity and affects both the affinity and conformation of the
interaction with DNA (Muscolini et al., 2009).
Tumour-associated mutations in TP53 are predominantly
point mutation (93,6%) that result in single amino-acid
substitutions - a mutational spectrum that is quite different from
that seen in other tumour-suppressor genes, in which large
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Cristina Piloto
deletions or frameshift mutations tend to result in the complete loss
of protein expression (Vousden and Lu, 2002).
The result of mutational inactivation of TP53 by singleamino-acid substituions is that many tumour cells retain the ability
to express the mutant p53 protein. These proteins are often more
stable than wild-type p53, and are present at very high levels in the
tumour cell. One explanation of this selection of such mutations is
that the mutant p53 proteins can act as dominant-negative
inhibitors of wild-type p53, which functions as a tetramer. The
observation that many tumours that harbour TP53 point mutations
also show loss of heterozygosity – effectively eliminating the wildtype allele – indicates that the efficiency of dominant-negative
inhibition might not be complete, and almostly certainly depends
on the nature of the initial point mutation (Vousden and Lu, 2002).
Tumour therapy that is based on the reactivation of p53 is
therefore an attractive goal, and might be achieved by reactivating
mutant p53, introducing exogenous wild-type p53 or activating
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endogenous p53 in those tumours that retain wild-type p53
(Vousden and Lu, 2002).
Furthermore, accumulating evidence that the apoptotic
activity of p53 is regulated separately from its other functions
indicate that it might be possible to selectively induce p53mediated apoptosis for cancer therapy (Vousden and Lu, 2002).
The abnormal proliferation, loss of normal cellular
environment and stress that accompanies malignant progression all
enhance the apoptotic sensivity of the cancer cells by either
increasing death signalling or hampering survival signals.
The suggestion that tumour cells show a greater propensity
to die in response to p53 than their normal counterparts has
triggered much excitement in the use of p53 as a tumour-specific
therapy (Vousden and Lu, 2002).
The group of Muscolini et al. has recently identified
mutation of Lys351 to Asn (K351N) as a new cancer-associated
mutant of p53 in a cisplatin-resistant clone derived from the A2780
ovarian carcinoma cell-line (A2780 CIS). This mutation was
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generated by the substitution AAG/AAT in heterozigosis.
Although K351N mutation did not affect p53 expression, it was
associated to the acquisition of resistance to apoptosis in
A2780CIS, particularly to defects in both CDDP-induced Bax
expression and associated mithocondrial membrane depolarization,
cytochrome C release and activation of caspase (Muscolini et al.,
2009).
Muscolini et al. found that K351N substitution did not
affect the overall half-life of p53 protein but significantly reduced
its ability to form tetramers, when compared to p53wt. Tetrameric
p53 is most effective at binding and transactivating p53 response
elements,
and
mutations
which
prevent
tetramerization
compromise DNA binding, diminish transactivation, and lead to
tumorigenesis (Friedman et al., 1993; Hainaut et al., 1994;
Halazonetis and Kandil, 1993; Hupp and Lane, 1994; Ishioka et
al., 1995; Lomax et al., 1998; Pietenpol et al., 1994; Tarunina et
al., 1996; Varley et al., 1996). As a consequence, p53K351N,
exhibited a reduced ability to transactivate specific target gene (Es.
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Bax, p53AIP1 and Δnp73) when overespressed in A2780 or Jurkat
cells. They also found that K351N substitution affected subcellular
localization of p53 by impairing its export from nucleus induced
by CDDP. These data identify K351N as a new cancer-associated
mutant in the TD of p53 with reduced tumor suupressor activity
and involved in the induction of resistance to drug-induced
apoptosis (Muscolini et al., 2009).
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AIM OF THE THESIS
The p53 tumor suppressor gene is a major barrier against
cancer, preventing tumor development and promotes apoptosis
induced by chemotherapy. P53 is able to regulate apoptosis both
through its transcriptional activity and by the induction of the outer
mitochondrial membrane (OMM) permeabilization promoting the
release of cytochrome c.
In the yeast S. cerevisiae, the machinery of the basic
apoptotic process seems to be conserved as it presents many of the
cytological markers of apoptosis such as chromatin condensation,
DNA fragmentation and the release of cytochrome c from
mitochondria to the cytoplasm. It was recently reported that the
expression of p53 in S. cerevisiae is able to activate the apoptotic
processes, making this system a good model to analyze the basic
mechanisms of apoptosis induced by p53.
It was recently identified a novel mutation in p53, in which
lysine 351 is replaced by an asparagine (K351N) in a cisplatin-
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resistant ovarian carcinoma cell line (A2780 CIS). The K351N
mutation is associated with the acquisition of resistance to
apoptosis induced by cis-platinum in this cell line.
The
K351N
mutation
significantly
reduces
the
thermodynamic stability of p53 tetramers, the transcriptional
activity of p53 and affects the export from the nucleus to the
cytosol induced by cisplatinum treatment. The characterization of
this mutant could thus help to envisage the development of new
cancer drugs that can suppress this phenotype in tumors.
We cloned the two human genes, p53 and its mutated form
p53K351N, under the control of Gal1-Gal10 vector and we
transformed them in both wild type and S. cerevisiae strains
mutated in genes involved in the apoptotic response. We then
analyzed the effect of the p53 expression during an apoptotic
stimulus (H2O2) and during aging.
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2. RESULTS AND DISCUSSION
2.1 EXPRESSION OF p53wt AND p53K351N IN
YEAST CELLS
To understand the involvement of p53 the and its mutated
form p53K351N in the phenomena of apoptosis and aging, we
have cloned these gene in the yeast pESC-HIS vector under the
control of Gal1-10 promoter. Gene expression is repressed in the
presence of glucose and induced by the presence of galactose in the
media. These vectors, were transformed in Saccharomyces
cerevisiae wild type (BY4741, see materials and methods) and
mutated strains in genes involved in the yeast apoptotic pathways
and mitochondrial morphology. In particular, we addressed our
attention to the following genes, also described in the introduction
sections 1.1.3-1.1.8:
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• YCA1: The YCA1 gene (YOR197w) encodes a
metacaspase that exhibits proteolytic activity homologous to
mammalian caspases (Madeo F. et al., 2002).
• AIF1: The yeast homologue AIF1 (YNR074c) encodes a
protein of 41.3 kDa, and shows 41% identity with human AIF.
• NUC1: NUC1 encodes for a yeast homologue of
mammalian EndoG, a mitochondrial nuclease that digests DNA
and RNA. Nuc1 has a role in both mitochondrial recombination,
apoptosis, and in the maintenance of polyploidy.
• NMA111: The protein Nma111, encoded by the
YNL123W ORF in yeast, is homologous to HtrA2(Omi) in
mammalian and, like all HtrA owns PDZ domains at the Cterminal (Pallene and Wren, 1997; Harris and Lim, 2001 ; Clausen
et al., 2002). They mediate protein-protein interactions involved in
signal transduction events. Δnma111 mutants show a lifespan
extension while the over expression of Nma111 promotes cell
death (Fahrenkrog et al., 2004).
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All these strains were also transformed, as a control, with
the empty plasmid (pESCHIS) and clones were selected on
minimal medium lacking histidine at 28 ° C.
The expression of p53 was verified by western blot analysis
(Figure 2.1.1). Protein extracts were prepared from cells grown in
minimal medium containing glucose (SD, promoter Gal repressed),
or after an incubation for about twenty hours in minimal medium
containing galactose (SGal) which induce Gal1-10 promoter and
induces the expression of p53 proteins.
Protein extracts were subjected to SDS-PAGE, transferred
to nitrocellulose membrane and hybridized with an antibody
directed against the p53.
As can be seen in figure 2.1.1, both p53 and p53K351N
were present only when cells are grown in the presence of
galactose, and not in glucose, suggesting that p53 cloned genes
were functional and produce proteins recognized by the p53
antibody.
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Figure 2.1.1 Western blot analysis of protein extracts from yeast strains
containing pESC-HIS-p53 (p53wt), pESC-HIS-p53K351N (p53K351N) and the
control vector p-ESC-HIS. Cells grown in minimal mediun containing glucose
(SD) were shifted for 20h in minimal mediun containing galactose (SG).
Ponceau red staining of membranes is also shown for quantization of transferred
proteins.
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Cristina Piloto
2.2 p53wt AND p53K351N INHIBIT THE GROWTH OF
YEAST CELLS
To explore the effect of p53wt and p53K351N on yeast cell
growth, we have performed a dilution spot assay on cells plated in
glucose, galactose, caffeine and glycerol. As shown in figure 2.1.4,
in the presence of glucose (SD), a condition that does not allow the
protein expression, there was no difference between strains
expressing the two forms of p53 and those expressing the empty
plasmid. On the contrary, in the presence of galactose (SGal)
growth of strains expressing both wt p53 and the K351N mutated
form was completely inhibited.
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Figure 2.2.1 . Dilution spot assay of the indicated strains on plates
containing 2% glucose (SD), 2%galactose (SGal), 3% glycerol (SY), 0,1%
caffeine. Plates were recorded after 3 days of incubated at 28°C or, where
indicated, at 37°C
The same experiment carried out at a temperature of 37 °C,
gave the same results, demonstrating that the protein inhibited cell
growth independently of growth temperature.
In the presence of glycerol, a respiratory carbon source, the
promoter (GAL1-10) is not suppressed, but results in a basal
transcription of genes located downstream. This low expression of
p53 genes resulted in the arrest of cell growth, suggesting that few
molecules of p53 are sufficient to block the cell cycle.
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Cristina Piloto
The presence of 0.10% caffeine inhibited more than 50%
cell growth in all tested conditions, so we can conclude that it did
not induce apoptosis in a p53 dependent manner.
We have performed the same experiment also with mutant
strains. As can be seen in figure 2.1.3, the expression of both
forms of p53 blocked the cell growth also when expressed in the
yca1 mutant strain. In fact, compared to growth on SD, which is
complete up to the fifth dilution, growth on galactose (sGAL) at
28°C was completely inhibited. This indicated that the expression
of protein blocks cell growth, despite the lack of the YCA1 gene,
which is involved in the apoptotic response. The growing
conditions are similar at 37°C, except when it is expressed
p53K351N, which determined the cell growth up to the first
dilution. This could be due to the fact that the mutated protein
defects in forming tetrameres increases with temperature and, in
absence of yca1, high levels of functional p53 protein are required
to block cell cycle.
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This can be also confirmed by the observation that cell
growth is only partially inhibited in the presence of glycerol
indicating that, at least in yca1 mutant strains, the basal expression
of p53 proteins is not sufficient for complete inhibition of cell
growth and over-expression is required.
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Figure 2.2.2 . Dilution spot assay of the indicated strains on plates
containing 2% glucose (SD), 2%galactose (SGal), 3% glycerol (SY), 0,1%
caffeine. Plates were recorded after 3 days of incubated at 28°C or, where
indicated, at 37°C
The presence of caffeine, in conjunction with the
expression of p53, did not completely inhibit cell growth, possibly
related to the lack of the gene YCA1.
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The partial resistance to the cell growth inhibition
following p53 expression was also observed in the aif1, nuc1 and
nma111 (Figure 2.1.3).
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Figure 2.2.3 . Dilution spot assay of the indicated strains on plates
containing 2% glucose (SD), 2%galactose (SGal), 3% glycerol (SY), 0,1%
caffeine. Plates were recorded after 3 days of incubated at 28°C or, where
indicated, at 37°C
The lack of growth in gycerol (SY) for aif1, nuc1 and
nma111 is due to the mitochondrial defects shown by these mutant
strains.
To explore the contribution of p53wt and p53K351N in
accumulation of reactive oxygen species (ROS), we have used the
fluorescence DHR123 labelling assay, which identify cells
containing ROS. These assays showed that cells transformed with
the plasmids containing p53wt and p53K351N have an
accumulation of intracellular ROS higher respect to cells
transformed with the empty plasmid pESC-HIS. In figure 2.2.4 are
shown a sample of cells ROS positive (left panel) and the
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quantification in percentage (right panel). About 35-40% of cells
expressing both forms of p53 were ROS positive, 3-4 fold more
than the same strain expressing the control plasmid p-ESC.
Figure 2.2.4 Cells containing p-ESC, p53wt and p53K351N were precultivated
in SD and then shifted for 20h in minimal mediun containing galactose to allow
the expression of p53 genes. DHR123 was added to the cultures at the
concentration of 5µg/ml and, after 3 hours at 28°C, cells were observed at the
fluorescence microscope (left panel). Quantification of ROS positive cell was
made in three independent experiments. In the right panel is shown the average
with SD.
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2.3 p53wt AND p53K351N INDUCE CELL DEATH WHEN
EXPRESSED IN YEAST CELLS
In order to study the effect of the expression of p53 and
p53K351N on cell viability and apoptosis, we compared the
percentage of viability, measured by clonogenicity, after twenty
hours from the expression of both protein forms in galactose
without (Figure 2.3.1) or in the presence of the apoptotic inducer
hydrogen peroxide (3mM H2O2 Figure 2.3.2).
The over-expression of both forms of p53 proteins resulted,
in the wild type strain, in a reduction of about 50% viability. The
reduction of viability was about 40% in yca1 and nma111 mutants.
In the case of aif1 and nuc1 mutants, the loss of viability was quite
low, suggesting that these strains are resistant to p53-induced cell
death.
Aif1 and Nuc1/EndoG are two mitochondrial proteins that
are released from this organelle upon an apoptotic stimulus. The
fact the aif1 and nuc1 mutants were more resistant, compared to
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the wild type, to p53 expression, suggests that Aif1 and Nuc1
proteins are involved in p53-promoted cell death. On the contrary,
p53-cell death is only partially dependent from active caspase
Yca1 or Nma111.
These results indicate that most of the p53 toxicity in yeast
is mediated by the association of p53 to the mitochondria. There
isn’t any evident difference between cell death induced by p53WT
and p53K351N, except for the aif1 and nuc1 strains, that seem,
especially nuc1, extremely resistant to the mutated form. As
p53K351N shows a reduced capability to tetramerize and to move
from the nucleus to cytoplasm, this mutated form would still
capable to induce cell death only in the presence of an active
Nuc1-EndoG protein.
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Figure 2.3.1 . Survival determined by clonogenicity of the indicated
strains overexpressing p53, p53K351 and vector control (p-ESC) yeast cells
after 20 hr induction on galactose. Average and standard deviation, obtained
from three independent experiments, are reported
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We then analyze cell viability in the presence of p53
proteins upon an apoptotic stimulus such as hydrogen peroxide
treatment. The comparison of cell viability after H2O2 treatment
(Figure 2.3.2) has first pointed out differences between mutant and
wild-type strains containing the plasmid control. In fact, all the
mutated strains have a higher resistance to hydrogen peroxide,
according to their role in mediating the apoptotic process.
The expression of p53 leads to increased sensitivity to apoptotic
stimuli, with the exception of nuc1 mutant in which is absent the
mammalian EndoG homologous nucleases.
In fact, cell viability of nuc1 mutant that expresses both
forms of p53 was similar to that shown by the control strain and is
very high compared with that of all other analyzed strains over
expressing the two forms of p53.
This result suggests that, at least in the yeast S. cerevisiae,
cell death induced by an apoptotic insult in the presence of p53 is
mediated through the action of Nuc1/EndoG.
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Figure 2.3.2 . Survival determined by clonogenicity of the indicated
strains overexpressing p53, p53K351 and vector control (p-ESC) yeast cells
after 20 hr induction on galactose + 4 h in 3mM H2O2. Average and standard
deviation, obtained from three independent experiments, are reported.
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2.4 EFFECTS OF p53wt AND p53K351N ON
YEAST CHRONOLOGICAL LIFESPAN
Long term cultivation causes an aging processes in the
whole yeast culture, called chronological aging (Fabrizio and
Longo, 2003), which leads to physiological-induced apoptosis in
yeast (Herker et al., 2004). Therefore, we investigated the effect of
p53wt and p53K351N in chronological aging in wild type and
mutant strains in genes involved in apoptosis. We observed that the
wild type strains BY4741 expressing p53wt and p53K351N, as
already discussed above, at the first day show a viability loss of
about 50%. Cell death kinetics was then very similar for both p53
and p53K351N expressing strains (Figure 2.4.1).
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Figure 2.4.1 . Chronologica aginng determined by clonogenicity of
BY4741 strains overexpressing p53, p53K351 and vector control (p-ESC).
Average and standard deviation, obtained from three independent experiments,
are reported.
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In the absence of the gene YCA1 (Figure 2.4.2) or NMA11
(Figure 2.4.3) cell death kinetics were slower compared to the wild
type. Anyway, also in these strains at the first day of p53
expression viability was approximately 50% .
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Figure 2.4.2 . Chronological aginng determined by clonogenicity of
yca1 strains overexpressing p53, p53K351 and vector control (p-ESC). Average
and standard deviation, obtained from three independent experiments, are
reported.
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Figure 2.4.3 . Chronological aginng determined by clonogenicity of
nma111 strains overexpressing p53, p53K351 and vector control (p-ESC).
Average and standard deviation, obtained from three independent experiments,
are reported.
The chronological aging of aif1 and nuc1 mutant strains
expressing the p53 proteins, showed that cells expressing the
mutated form p53K351N had a cell death kinetics very similar to
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that of the same strain expressing the control plasmid p-ESC
(figure 2.4.4 and 2.4.5).
These results are in agreement with those described in the
above paragraph and point to Aif1 and Nuc1 as the principles
mediator of cell death upon p53 expression.
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Figure 2.4.4 . Chronological aginng determined by clonogenicity of
nuc1 strains overexpressing p53, p53K351 and vector control (p-ESC). Average
and standard deviation, obtained from three independent experiments, are
reported.
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Figure 2.4.5 . Chronological aginng determined by clonogenicity of
nuc1 strains overexpressing p53, p53K351 and vector control (p-ESC). Average
and standard deviation, obtained from three independent experiments, are
reported.
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2.5 STUDY OF MITOCHONDRIAL MORPHOLOGY UPON
p53 AND p53K351N EXPRESSION
It has been recently reported that the functional
transcription-independent p53 apoptotic functions and p53induced mitochondria fragmentation are conserved in yeast
(Abdelmoula-Souissi et al, 2011; Coutinho et al, 2011).
The recombinant strains BY4741/pESC (negative control),
BY4741/p53WT (expressing wt p53) and BY4741/p53K351N
were transformed with a plasmid encoding the GFP targeted to the
mitochondria
allowing
visualization
of
the
mitochondrial
morphology (Westermann and Neupert 2000). Cells were
cultivated in SD and shifted in SGal for 20h to allow the
expression of p53. As illustrated in Figure 2.5.1, panel A, cells
expressing p53 presented fragmented mitochondria, whereas in
those expressing the p53K351N the percentage of aberrant
mitochondria was much lower. The panel B in figure 2.5.1
represents the quantification of glucose (SD) or galactose (SGal)
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grown cells presenting fragmented mitochondria. It is evident that
p53K351N mutated protein was not able to accumulate to
mitochondria and promote its fragmentation (Muscolini et al., 2011
and Dott. Palermo personal communication).
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Picture 2.5.1
K351N mutation affects p53-induced fragmentation of
mitochondria in S. cerevisiae. The BY4741 S. cerevisiae strain cells containing
pEsc-His, p53WT, and p53K351N, were all transformed with mito-GFP.
Transformants were grown in glucose (SD), and then shifted for 21 h in minimal
medium containing galactose (SGal). The mitochondria morphology (A) was
observed by analyzing the GFP with an Axioskop2 fluorescence microscope
(Carl Zeiss, Jena, Germany) equipped with a digital camera (micro-chargecoupled device). The quantification of cell population showing fragmented
mitochondria is reported in B. Data represent the mean and SD of three
independent experiments.
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3. CONCLUSIONS
The p53 phosphoprotein is probably the best known of all
tumor suppressors. It is well established that loss or inhibition of
p53 prevents cellular senescence and apoptosis. p53 is either lost or
mutated in about half of all human cancers.
Muscolini et al. found that K351N substitution did not
affect the overall half-life of p53 protein but significantly reduced
its ability to form tetramers, when compared to p53wt. They also
found that K351N substitution affected subcellular localization of
p53 by impairing its export from nucleus induced by CDDP. These
data identify K351N as a new cancer-associated mutant in the TD
of p53 with reduced tumor suupressor activity and involved in the
induction of resistance to drug-induced apoptosis (Muscolini et al.,
2009).
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The characterization of this mutant could thus help to
envisage the development of new cancer drugs that can suppress
this phenotype in tumors.
In the yeast S. cerevisiae, the machinery of the basic
apoptotic process seems to be conserved as it presents many of the
cytological markers of apoptosis such as chromatin condensation,
DNA fragmentation and the release of cytochrome c from
mitochondria to the cytoplasm. It was recently reported that the
expression of p53 in S. cerevisiae is able to activate the apoptotic
process, making this system a good model to analyze the basic
mechanisms of apoptosis induced by p53.
The data obtained in this thesis, have highlighted that the
over-expression of wild-type p53 and p53K351N proteins in yeast
blocks cell cycle and reduces viability.
We noticed the loss of viability of about half of the cell
population, suggesting that the over expression of p53 may play a
role in removing old or damaged cells of the cell population.
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This effect, pointed out in wild yeast strains, was attenuated
in strains mutated in genes encoding component of the yeast
apoptotic pathway. In fact, a complete block of cell cycle in yca1
mutant can be achieved only when p53 are over-expressed a at
37°C only the p53 wild type is able to inhibit cell growth. The
strains that showed the higher resistance to p53 expression were
aif1 and nuc1, indicating that Aif1 and Nuc1 (EndoG) are the
major proteins mediating p53-induced cell death in yeast. The p53
expression rendered cells more susceptible to cells death induced
by H2O2, an apoptotic inducer, except for the nuc1 mutant that
showed very little difference in cell viability of strains expressing
both forms of p53 and the control plasmid, pointing to Nuc1 as an
important protein mediating cell death induced by H2O2 in the
presence of p53.
The aif1 and nuc1 mutants, in particularly the latter one,
were also highly resistant to the mutated form p53K351N,
measures both after 20h of gene expression induction and during
chronological aging. Upon p53 over-expression we also observed
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an increase in mitochondrial fragmentation, a phenomenon greatly
reduced for the mutated K351N form. We can speculate that, in
yeast cells, the expression of p53, beside its transcriptional effect,
cause mitochondrial fragmentation that release the proapoptotic
factors Aif1 and Nuc1. In the absence of these proteins this effect
is attenuated and, when mitochondrial fragmentation does not
occur (K351N) cell death is not completely induced.
On the contrary, the yeast caspase Yca1 and the Nma111
(Omi homologue), seem marginally involved in mediating p53
toxicity.
In the future we plan to explore more p53 functions through
the use of multiple mutants involved in mitochondrial morphology
and post-translational modifications linked to the p53 cytoplasmic
function.
The genetic tractability of budding yeast, its ease of
manipulation and the wealth of functional genomics tools available
will be very useful to isolate proteins involved in this particular
function of p53, a protein so important in human disease.
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4. MATHERIALS AND METHODS
Transformation of yeast cells with lithium acetate
Exponentially growing cells were collected at the
concentration of 2x107 cells / ml, collected by centrifugation,
washed once with sterile water and then washed with 1 ml of 100
mM lithium acetate.
After centrifugation, cells were incubated with a reaction
mixture composed of:
240 µl PEG (50% w / v)
36 µl 1.0 M Liac
50 l SS-DNA (2.0 mg / ml)
plasmid DNA (0.1-1.0 g)
34-µl sterile dd H2O
After incubation at 28 ° C for 30 min, cells were heat
shocked at 42 ° C for 30 min., centrifuged, resuspended in 1 ml of
sterile H2O and plated on selective medium.
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Cell viability determination
Cells were grown at 28 °C in SD medium (yeast nitrogen
base) containing auxotrophic requirements as needed. 5 µl of cell
suspensions containing approximately 6·106 cells / ml were poured
on a thin layer of YPD agar on a microscope slide. A cover slip
was placed over the samples and, after 24 h, viable and unviable
cells were scored on the basis of their ability to form
microcolonies.
H2O2 sensitivity determination
Exponentially growing cells were treated with 0.8, 1.2 and
3 mM H2O2 for 4h at 28°C . After treatments, cell viability was
measured as described above.
Fluorescence microscopy
The presence of ROS was detected with DHR (Sigma
Aldrich Co. D1054), as described previously (Madeo et al,1999).
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Free 30-OH ends were detected by TUNEL on cells grown for 9
days, as described (Madeo et al, 1997).
Phenotypes determination by microtiter analysis
Cells were grown at 28 °C in SD medium additioned with
auxotrophic requirements. 5 µl of 10-fold dilutions were the
spotted on solid SD medium supplemented, as specified in the text,
with 2% galactose (S-Gal), 3% glycerol (SY) 0,10% caffeine
(SD+caffeine) or temperature at 37° C. Growth of colonies was
observed after incubation of plates for 3 days at 28 °C .
Protein extracts, SDS-PAGE and western blot analysis
Exponential and stationary phase cells
Cell extracts were prepared from culture growing at 28 °C
in shaked flasks containing SD medium with the addition of
auxotrophic requirements. Exponential cells were collected at 0.2
OD600 density while stationary cells were grown for two
additional days.
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Samples were centrifuged at 4 °C and washed with water.
Cells were resuspended with 0.2 ml of 2M NaOH, 5% βmercaptoethanol and incubated in ice for 10 min. After the addition
of 40 ml of 50% trichloro acetic acid, samples were incubated in
ice for further 10 min. After centrifugation, cell pellets were dried
at room temperature for 5 min and resuspended in lysis buffer
(50mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 2% SDS, 0.1%
bromophenol blue, 10% glycerol).
Proteins in the extracts were separated by standard SDSpolyacrylamide gel electrophoresis procedures.
Detection of proteins by western blot analysis
After
SDS-polyacrylamide
gel
electrophoresis
fractionation, proteins were transferred following standard
protocols onto commercial membrane. The protein p53 was
detected by hybridization with the antibody goat anti-mouse
SC126 and goat anti-mouse.
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Signals were revealed with Super Signal West Pico
chemiluminescent substrate HRP (Pierce).
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RINGRAZIAMENTI
Nel completare questo mio percorso di studi desidero
ringraziare la mia relatrice, Cristina Mazzoni, che mi ha seguito
scrupolosamente nel corso del dottorato di ricerca, tutte le persone
che ho incontrato in questi tre anni, Vanessa Palermo, Eleonora
Mangiapelo, Francesco Giorgi, Federica Straforini, Luisa Pieri,
Patrizia Luise, Michele Saliola, Michela Pitorri, tutti coloro che
sono passati per il lab 25, tutti i “residenti” del piano rialzato, e
tutti gli altri dottorandi che stanno raggiungendo con me questo
traguardo .
Ringrazio il Prof. Claudio Falcone e tutti gli altri docenti e
ricercatori che sono stati presenti nel corso delle mie giornate
lavorative in questi tre anni, e quelli che mi hanno invece guidato
verso la laurea.
Desidero ringraziare anche i miei vecchi compagni di
studi, che hanno proseguito con me, anche se in modi diversi, i
loro percorsi formativi.
Pag. 141
Cristina Piloto
Un ringraziamento particolare va al dott. Carlo Anzilotti e
a tutte le persone che insieme a me stanno lavorando per
conquistarsi un’identità sempre più bella e solida, e che permetta
di raggiungere obiettivi concreti..
Ringrazio tutte le persone che mi stanno vicino, e che
credono in me e nelle mie capacità.
Pag 142
Dottorato di Ricerca in Biologia Cellulare e dello Sviluppo
Pag. 143