Transcript 13.2. Natural Cell Death

Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Zoltan Balajthy
Molecular Therapies- Lecture 13
CELL CYCLE AND
CANCER, P53
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Learning objectives of chapter 12 and 13 . The purpose of this chapter is to describe the processes and
regulations of both cell cycle and cell death, explain the unregulated cell division, and to point out the
therapeutic intervention in cancer at molecular levels.
Topics in chapter 13.
13.1. Tumor suppressor genes, and their biochemical functions
The retinoblastoma protein
Primary structure of transcription factor p53 and its regulation
Restoration of p53 function in tumor cells
13.2. Natural Cell Death
Common elements of the three forms of natural cell death
Biochemical pathways of caspase activation dependent cell death
Killing tumours by induction of apoptosis
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Transcriptional Events in G1 Phase of Cell-cycle
CDK inhibitors
Start of S phase
DNS replication
machinery
pozitív
erősítés
Dihydrofolat reductase
Thymidine kinase
Thymidylate synthase
DNA polymerase
CDK inhibitors
E2F: transcription factor E2F1
EGF: epidermal growth factor
CDK: cyclin-dependent protein kinase
Rb: retinoblastoma protein
D1, A, E: Cyclin D1, A és E
transzkripció
leállítás
DNS replication
machinery
CDK inhibitors
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Tumor Suppressor Genes, and Their Biochemical Functions
Name
Chromosomal localizationsBiochemical function of missing protein
p53
17
induces CDK inhibitor p21, induces GADD45 which induces
DNS repair, induces apoptosis
NF-1
17
Neurofibromatosis, type-1
neurofibromine (activation of ras GTPase)
WT- 1 (Wilms-tumor)
11
four Zn-finger transcription factor
APC
adenoma polyposis coli
5
induction of apoptosis, interacts with β-catenin
P16 melanoma
9
inhibitor of cdk4
PTEN
deleted in prostate cancer
P1 phosphatase
BRCA1
breast cancer
17
DNS repair
BRCA1
Breast cancer
13
DNS repair
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The Retinoblastoma Example
The retinoblastoma gene
180 kb, 27 exon
4.7 kb mRNA
105 kD fehérje
Deletion was observed in this
gene when observed from isolated
tumor cells.
The frequency of deletions in
this genes corresponded
to the rate of occurrence of
of this tumor. From neuroblastoma
tumor cells only damaged or mutated
forms of this gene could be isolated.
Re-introducing the cloned Rb gene
into the tumor cells led to their normal
proliferation (loss of tumor forming
Potential)
Some part of chromosome 13 were very often
missing when it was isolated from neuroblastoma
tumors. From the corresponding part in normal
chromosome 13 the neuroblastoma gene could
be cloned and characterized.
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Tumor Suppressor Genes: Retinoblastoma and P53
Rb
Blockade of
cell divisin
Damage
to DNA
E2F
G1 CDK
active
p
p
Rb
p
P53
p
TF
E2F
Ac
1. Halts cell cycle
at G1 csheckpoit
2. Activates DNA
repair system
Ac
S phase / G2
Initiates transcription
of p21
p21
Mithosis
p21
Cell Division
Blocks cell cycle
at G1 checkpoin
G1 CDK
active
Initiates transcription
of repair enzymes
DNA repair
Prevents DNA
replication
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Regulation Transcription Factor of p53 I.
DNA Damage, UV
Oncogenic Stimulation
Hypoxia
Hypoglycemia
(cyclins, c-Myc, E2F-1, Ras)
BRCA1
(ATM, DNA-PK, ATR)
p19ARF
The p53 transcription factor can either induce growth
arrest or apoptosis in response to a variety of
cellular stresses including exposure to DNA
damaging agents, hypoxia and inappropriate
proliferative signals. DNA damaging agents and
UV irradiation stabilize p53 through phosphorylation
of p53 at its N-terminal and activate its DNA binding
through dephosphorylation and acetylation of its
C-terminal region. Hypoxia and hypoglycemia
stabilize p53 through both phosphorylation
dependent and independent mechanisms.
Inappropriate oncogene stimulation leads to
p53 stabilization through the p19ARF pathway.
Binding of hdm2 to p53 inhibits its transactivation
activity and leads to its degradation.
ARF overexpression leads to p53 stabilization
by binding to hdm2 and preventing the hdm2
mediated p53 inhibition and degradation.
Disruption of hdm2 and p53 interactions
appears to be critical for the stabilization of p53.
Stabilized and activated p53 can then transactivate
its target genes.
HIF-1
h
d
m
2
?
p
p53
h
d
m
2
p53
acetylation
dephosphorylation
Stable
Degraded p53
p53 p53
p53 p53
Stable and Active
Transcriptional
repression
Transcription of target genes
IAP
survivin
PUMA
NOXA
APAF-1
P21(WAF1)
GADD45 14-3-3
PIG3
BAX FAS/APO1 KILLER/DR5
cell death
proteins
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Regulation Transcription Factor of p53 II.
DNA damage
hdm2
ubiquitin
ATM / ATR
Chk2
p300
p300
Hmd2
p53
Hmd2
p p53
p p p53
Hmd2
p53 destruction
p53 stabilization
and tetramerization
gene expression
p53 turnover
in normal conditions
Cell cycle arrest
Cell death
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Primary Structure of Transcription Factor p53
ATM/ATR
p
p
Hdm2
p300
Chk2
Ser Ser
15
20
ubiquitination or
acetylation
Lys Lys Lys Lys
372 381
hdm2
1
382 383
nuclear export signal
100
200
300
393
N
transcriptional
activation domain
C
Sequence-specific DNA-binding
domain
tetramerization
domain
p53 is of central importance in the response to DNA damage and other cellular stresses,
and its activation can cause the death of the cell. It is therefore subject to an unusually
large array of regulatory modifications that ensure it is present and active only when
necessary. Most of these modifications increase its concentration or its intrinsic gene
regulatory activity, or both, when DNA damage occurs
Mutation of the gene for ATM in humans results in the disease ataxia telangiectasia,
which is characterized by, among other things, a greatly reduced ability to repair
radiation-induced double-strand breaks – and an increased risk of developing cancer.
ATM is recruited to sites of double-strand break formation, where it phosphorylates
effector molecules that carry out the damage response.
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Restoration of p53 Function in Tumor Cells III.
Cell stress
Oncogene activity
Prima-1
CP-31398
Cell death
Hdm2
p53
Growth arrest
Arf
Nutlins
Nutlins act by blocking interaction of Mdm2 with p53 , therefore prevents
its destruction leading to more of the the stable form of p53
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Restoration of p53 Function in Tumor Cells II.
DNA Damage, UV
Oncogenic Stimulation
Hypoxia
Hypoglycemia
(cyclins, c-Myc, E2F-1, Ras)
BRCA1
(ATM, DNA-PK, ATR)
p19ARF
HIF-1
?
The p53 transcription factor
p
h
acts as a tumor suppressor Oncogenic
d
by inducing growth arrest or mutations
m
p53
2
apoptosis in response to a
variety of cellular stresses
Nutlin
therapy
including DNA damage,
hypoxia and inappropriate
p53
acetylation
dephosphorylation
proliferative signals.
Stable
Gene therapy
Stabilizing molecules
h
d
m
2
Degraded p53
p53 p53
p53 p53
Stable and Active
Transcriptional
repression
Transcription of target genes
IAP
survivin
PUMA
NOXA
APAF-1
P21(WAF1)
GADD45 14-3-3
PIG3
KILLER/DR5
FAS/APO1
BAX
cell death
proteins
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13.2. Natural Cell Death
is a physiologic phenomenon occurring continuously in living tissues to remove cells
without any function (morphogenesis, duplicate structures, sexual dimorphism),
which are produced in excess (e.g. in bone marrow), which develop improperly (e.g.
part of lymphocytes), which completed their function (endometrium, tissue turnover),
which are potentially dangerous (e.g. autoreactive T cells, neutrophil granulocytes).
Forms of natural cell death
a. Programmed cell death
Embryogenesis
functional, developmental definition; predictable in space and time;
requires active protein synthesis
b. Specialized forms of cell death
tissue-specific terminal differentiation; the death program is suspended at one point
of the death pathway; the partial death forms serve specific tissue functions; requires
active protein (e.g., red blood cell, platelets, lens, cornification)
c. Apoptosis
Morphologic definition
can be induced by non-physiologic agents
does not always require active protein synthesis
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Common Elements of The Three Forms of Natural Cell Death
elimination of the nucleus
DNA degradation by endonucleases acting at internucleosomal sites
activation and/or induction of protein cross-linker transglutaminases
Activation of specific proteases
there is no leakage of intracellular macromolecules
effective phagocytosis with the help of integrin receptors
(except cornification and lens epithelial cells)
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Morphology of Apoptosis
Condensation
Separation
Fragmentation
Phagocytosis
Residual
body
Lysosomal
digestion
‘HISTIOCYTE’
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Biochemical Pathways of Caspase Activation Dependent Cell Death
Mitochondrion
Plasma membrane
Truncated
Bid
Bcl-2, …
Bid
Cytochrome c
Procaspase-3
Procaspase-8
ATP
Bax, Noxa, Puma, …
Apaf-1
Procaspase-9
Caspase-8
p53
Mdm2
Apoptosome
Caspase-3
XIAP
Diablo/SMAC
Cell death
Cytoplasma
Nucleus
Prot
e
deat olysis o
h su
bstra f
tes
DNA
cleavage
ICAD
CAD
DNA-PK,
ATM
DNA damage
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Hystology staining
Apoptosis staining
Killing Tumours by Induction of Apoptosis
Untreated control
Radiotherapy (RT)
Deathl ligand
(TRAIL)
Radiotherapy (RT)
+
Death ligand (TRAIL)
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