Cell Cycle and Cancer , p53
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Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
CELL CYCLE AND CANCER, P53
Zoltan BalajthyMolecular Therapies- Lecture 13
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
Topics in chapter 13.
13.1. Tumor suppressor genes, and their biochemical functionsThe retinoblastoma proteinPrimary structure of transcription factor p53 and its regulationRestoration 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 deathKilling tumours by induction of apoptosis
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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.
CDK inhibitors
CDK inhibitors
CDK inhibitors
Dihydrofolat reductaseThymidine kinase Thymidylate synthase DNA polymerase
E2F: transcription factor E2F1 EGF: epidermal growth factorCDK: cyclin-dependent protein kinaseRb: retinoblastoma proteinD1, A, E: Cyclin D1, A és E
DNS replication machinery
Start of S phase
pozitív erősítés
transzkripcióleállítás
Transcriptional Events in G1 Phase of Cell-cycle
DNS replication machinery
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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 neurofibromine (activation of ras GTPase)Neurofibromatosis, type-1
WT- 1 (Wilms-tumor) 11 four Zn-finger transcription factor
APC 5 induction of apoptosis, interacts with β-cateninadenoma polyposis coli
P16 melanoma 9 inhibitor of cdk4
PTEN P1 phosphatasedeleted in prostate cancer
BRCA1 17 DNS repairbreast cancer
BRCA1 13 DNS repairBreast cancer
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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)
The Retinoblastoma Example
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
Cell Division
1. Halts cell cycleat G1 csheckpoit
2. Activates DNArepair system
Damageto DNA
P53
Initiates transcriptionof p21
Initiates transcriptionof repair enzymes
DNA repair
p21
G1 CDKactive
p21Blocks cell cycleat G1 checkpoin
Prevents DNAreplication
E2FRb
Ac
AcE2F
G1 CDKactive
TF
Rbp p
pp
S phase / G2
Mithosis
Blockade ofcell divisin
Cell Division
1. Halts cell cycleat G1 csheckpoit
2. Activates DNArepair system
Damageto DNA
P53
Initiates transcriptionof p21
Initiates transcriptionof repair enzymes
DNA repair
p21
G1 CDKactive
p21Blocks cell cycleat G1 checkpoin
Prevents DNAreplication
1. Halts cell cycleat G1 csheckpoit
2. Activates DNArepair system
1. Halts cell cycleat G1 csheckpoit
2. Activates DNArepair system
Damageto DNA
P53
Initiates transcriptionof p21
Initiates transcriptionof repair enzymes
DNA repair
p21p21
G1 CDKactive
p21p21Blocks cell cycleat G1 checkpoin
Prevents DNAreplication
E2FRb
Ac
AcE2F
G1 CDKactive
TF
Rbp p
pp
S phase / G2
Mithosis
Blockade ofcell divisin
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Oncogenic Stimulation DNA Damage, UV
hdm2
hdm2
p53
p53
p
BRCA1
(cyclins, c-Myc, E2F-1, Ras)
p19ARF
HypoxiaHypoglycemia
HIF-1?
(ATM, DNA-PK, ATR)
Degraded p53
Stable and Active
Transcription of target genes PUMA
P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5PIG3
APAF-1NOXA
Stable
acetylationdephosphorylation
Transcriptionalrepression IAP
survivin
cell death proteins
p53 p53
p53p53
Oncogenic Stimulation DNA Damage, UV
hdm2
hdm2
p53
p53
p
BRCA1
(cyclins, c-Myc, E2F-1, Ras)
p19ARF
HypoxiaHypoglycemia
HIF-1?
(ATM, DNA-PK, ATR)
Degraded p53
Stable and Active
Transcription of target genes PUMA
P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5PIG3
APAF-1NOXA
Stable
acetylationdephosphorylation
Transcriptionalrepression IAP
survivin
cell death proteins
p53 p53
p53p53
hdm2
hdm2
p53
p53
p
BRCA1
(cyclins, c-Myc, E2F-1, Ras)
p19ARF
HypoxiaHypoglycemia
HIF-1?
(ATM, DNA-PK, ATR)
Degraded p53
Stable and Active
Transcription of target genes PUMA
P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5PIG3
APAF-1NOXA
Stable
acetylationdephosphorylation
Transcriptionalrepression IAP
survivin
cell death proteins
p53p53 p53p53
p53p53p53p53
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.
Regulation Transcription Factor of p53 I.
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Hmd2 p53 p53pp p53p
p300
p300
DNA damage
ATM / ATR
Chk2
Hmd2
Hmd2
gene expression
Cell cycle arrest Cell death
p53 destruction p53 stabilization and tetramerization
ubiquitin
Regulation Transcription Factor of p53 II.
hdm2
p53 turnover in normal conditions
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transcriptionalactivation domain
Sequence-specific DNA-bindingdomain
tetramerizationdomain
CN1 100 200 300 393
nuclear export signal15 20
Ser Ser381 382Lys Lys
372Lys
383Lys
Hdm2 ubiquitination orp300 acetylation
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.
Primary Structure of Transcription Factor p53
p p
ATM/ATR
Chk2
hdm2
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Cell stressOncogene activity
p53 Hdm2 Arf
Nutlins
Prima-1CP-31398
Cell death Growth arrest
Nutlins act by blocking interaction of Mdm2 with p53 , therefore prevents its destruction leading to more of the the stable form of p53
Restoration of p53 Function in Tumor Cells III.
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hdm2
hdm2
p53
p53
p
BRCA1
Oncogenic Stimulation(cyclins, c-Myc, E2F-1, Ras)
p19ARF
HypoxiaHypoglycemia
HIF-1?
DNA Damage, UV
(ATM, DNA-PK, ATR)
Degraded p53
Stable and Active
Transcription of target genes PUMA
P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5PIG3
APAF-1NOXA
Stable
acetylationdephosphorylation
Transcriptionalrepression
IAPsurvivin
cell death proteins
p53 p53
p53p53
The p53 transcription factor acts as a tumor suppressor by inducing growth arrest or apoptosis in response to a variety of cellular stresses including DNA damage, hypoxia and inappropriate proliferative signals.
Oncogenicmutations
Gene therapyStabilizing molecules
Nutlintherapy
hdm2
hdm2
hdm2
hdm2
p53
p53
pp
BRCA1
Oncogenic Stimulation(cyclins, c-Myc, E2F-1, Ras)
p19ARF
HypoxiaHypoglycemia
HIF-1?
DNA Damage, UV
(ATM, DNA-PK, ATR)
Degraded p53
Stable and Active
Transcription of target genes PUMA
P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5PIG3
APAF-1NOXA
Stable
acetylationdephosphorylation
Transcriptionalrepression
IAPsurvivin
cell death proteins
p53 p53
p53p53
p53p53 p53p53
p53p53p53p53
The p53 transcription factor acts as a tumor suppressor by inducing growth arrest or apoptosis in response to a variety of cellular stresses including DNA damage, hypoxia and inappropriate proliferative signals.
Oncogenicmutations
Gene therapyStabilizing molecules
Nutlintherapy
Restoration of p53 Function in Tumor Cells II.
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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 deatha. Programmed cell death Embryogenesisfunctional, developmental definition; predictable in space and time;requires active protein synthesis
b. Specialized forms of cell deathtissue-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 definitioncan be induced by non-physiologic agentsdoes 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
Separation
Condensation
Fragmentation
Phagocytosis
LysosomaldigestionResidual
body
‘HISTIOCYTE’
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Biochemical Pathways of Caspase Activation Dependent Cell Death
Caspase-8
Pro-caspase-8
NucleusNucleus
Cytochrome c
Apaf-1Pro-
caspase-9
ATP
Apoptosome
Diablo/SMACXIAP
Caspase-3
Bid
TruncatedBid
DNAcleavage
Pro-caspase-3
Mitochondrion
DNA damageDNA damage
DNADNA--PK, PK, ATMATM
Mdm2p53
Bax, Noxa, Puma, …
CAD
Bcl-2, …
ICADICAD
Cell death
Plasma membrane
Cytoplasma
Caspase-8
Pro-caspase-8
NucleusNucleus
Cytochrome c
Apaf-1Pro-
caspase-9
ATP
Apoptosome
Diablo/SMACXIAP
Caspase-3
Bid
TruncatedBid
DNAcleavage
Pro-caspase-3
Mitochondrion
DNA damageDNA damage
DNADNA--PK, PK, ATMATM
Mdm2p53
Bax, Noxa, Puma, …
CAD
Bcl-2, …
ICADICAD
Cell death
Plasma membrane
Cytoplasma
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Killing Tumours by Induction of Apoptosis
Untreated control Radiotherapy (RT) Deathl ligand (TRAIL)
Radiotherapy (RT) +
Death ligand (TRAIL)
Apo
ptos
is s
tain
ing
Hys
tolo
gy s
tain
ing
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TÁMOP-4.1.2-08/1/A-2009-0011
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