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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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.

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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.

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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.

TÁMOP-4.1.2-08/1/A-2009-0011

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.

TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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)

TÁMOP-4.1.2-08/1/A-2009-0011

Morphology of Apoptosis

Separation

Condensation

Fragmentation

Phagocytosis

LysosomaldigestionResidual

body

‘HISTIOCYTE’

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

TÁMOP-4.1.2-08/1/A-2009-0011