at the University of Pécs and at the University of Debrecen Identification number

(1)

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

(2)

CELL CYCLE AND CANCER, P53

Zoltan Balajthy

Molecular Therapies- Lecture 13

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

(3)

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

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.

(4)

CDK inhibitors

CDK inhibitors CDK inhibitors

Dihydrofolat reductase Thymidine kinase Thymidylate synthase DNA polymerase

E2F: transcription factor E2F1 EGF: epidermal growth factor

CDK: cyclin-dependent protein kinase Rb: retinoblastoma protein

D1, A, E: Cyclin D1, A és E

DNS replication machinery

Start of S phase

pozitív erősítés

transzkripció leállítás

Transcriptional Events in G1 Phase of Cell-cycle

DNS replication

machinery

(5)

Tumor Suppressor Genes, and Their Biochemical Functions

Name Chromosomal localizations Biochemical 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-catenin

adenoma polyposis coli

P16 melanoma 9 inhibitor of cdk4

PTEN P1 phosphatase

deleted in prostate cancer

BRCA1 17 DNS repair

breast cancer

BRCA1 13 DNS repair

Breast cancer

(6)

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.

(7)

Tumor Suppressor Genes: Retinoblastoma and P53

Cell Division

1. Halts cell cycle at G1 csheckpoit

2. Activates DNA repair system

Damage

to DNA

P53

Initiates transcription of p21

Initiates transcription of repair enzymes

DNA repair p21

G1 CDK active

p21 Blocks cell cycle

at G1 checkpoin

Prevents DNA replication

E2F Rb

Ac

E2F

Ac G1 CDK

active

TF

p Rb p

p p

S phase / G2

Mithosis

Blockade of

cell divisin

Cell Division

1. Halts cell cycle at G1 csheckpoit

Damage

to DNA

P53

p21 Blocks cell cycle

at G1 checkpoin

Prevents DNA replication 1. Halts cell cycle

at G1 csheckpoit

2. Activates DNA repair system 1. Halts cell cycle

at G1 csheckpoit

Damage

to DNA

P53

p21

p21 p21 Blocks cell cycle

at G1 checkpoin

Prevents DNA replication

E2F Rb

Ac

E2F

Ac G1 CDK

active

TF

p Rb p

p p

S phase / G2

Mithosis

Blockade of

cell divisin

(8)

Oncogenic Stimulation DNA Damage, UV

h d m 2

h d m 2 p53

p53

p

BRCA1

(cyclins, c-Myc, E2F-1, Ras)

p19ARF

Hypoxia Hypoglycemia

HIF-1 ?

(ATM, DNA-PK, ATR)

Degraded p53

Stable and Active

Transcription of target genes PUMA

P21(WAF1) GADD45 14-3-3 BAX FAS/APO1KILLER/DR5 PIG3

APAF-1 NOXA

Stable

acetylation dephosphorylation

Transcriptional

repression _survivin^IAP

cell death proteins p53 p53

p53 p53

Oncogenic Stimulation DNA Damage, UV

h d m 2

h d m 2 p53

p53

p

BRCA1

p19ARF

HIF-1 ?

Degraded p53

APAF-1 NOXA

Stable

Transcriptional

p53 p53 h

d m 2

h d m 2 p53

p53

p

BRCA1

p19ARF

HIF-1 ?

Degraded p53

APAF-1 NOXA

Stable

Transcriptional

cell death proteins p53

p53 p53p53 p53 p53 p53 p53 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.

(9)

Hmd2 p53 p p p53 p p53

p300 p300

DNA damage

ATM / ATR

Chk2

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

(10)

transcriptional activation domain

Sequence-specific DNA-binding domain

tetramerization domain

C N

1 100 200 300 393

nuclear export signal 15 20

Ser Ser

381 382

Lys Lys

372

Lys

383

Lys Hdm2 ubiquitination or

p300 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

(11)

Cell stress Oncogene activity

p53 Hdm2 Arf

Nutlins Prima-1

CP-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.

(12)

h d m 2

h d m 2 p53

p53

p

BRCA1

Oncogenic Stimulation

p19ARF

HIF-1 ?

DNA Damage, UV

Degraded p53

P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5 PIG3

APAF-1 NOXA

Stable

Transcriptional

p53 p53

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.

Oncogenic mutations

Gene therapy Stabilizing molecules

Nutlin therapy

h d m 2 h d m 2

h d m 2 h d m 2 p53

p53

p p

BRCA1

Oncogenic Stimulation

p19ARF

HIF-1 ?

DNA Damage, UV

Degraded p53

P21(WAF1) GADD45 14-3-3 BAX FAS/APO1 KILLER/DR5 PIG3

APAF-1 NOXA

Stable

Transcriptional

p53 p53 p53 p53 p53p53

p53 p53 p53 p53

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.

Oncogenic mutations

Gene therapy Stabilizing molecules

Nutlin therapy

Restoration of p53 Function in Tumor Cells II.

(13)

(14)

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

(15)

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)

(16)

Morphology of Apoptosis

Separation

Condensation

Fragmentation

Phagocytosis

Lysosomal digestion Residual

body

‘HISTIOCYTE’

(17)

Biochemical Pathways of Caspase Activation Dependent Cell Death

Caspase-8

Pro- caspase-8

Nucleus

Nucleus Cytochrome c

Apaf-1

Pro- caspase-9

ATP

Apoptosome

Diablo/SMAC XIAP

Caspase-3

Bid

Truncated

Bid

DNA cleavage

Pro- caspase-3

Mitochondrion

DNA damage DNA damage

DNA

DNA- -PK, PK, ATM

ATM

p53

Mdm2

Bax, Noxa, Puma, …

CAD

Bcl-2, …

ICAD ICAD

Cell death

Proteoly

sis of death substra

tes

Plasma membrane

Cytoplasma Caspase-8

Pro- caspase-8

Nucleus

Nucleus Cytochrome c

Apaf-1

Pro- caspase-9

ATP

Apoptosome

Diablo/SMAC XIAP

Caspase-3

Bid

Truncated

Bid

DNA cleavage

Pro- caspase-3

Mitochondrion

DNA damage DNA damage

DNA

DNA- -PK, PK, ATM

ATM

p53

Mdm2

Bax, Noxa, Puma, …

CAD

Bcl-2, …

ICAD ICAD

Cell death

Proteoly

sis of death substra

tes

Plasma membrane

Cytoplasma

(18)

Killing Tumours by Induction of Apoptosis

Untreated control Radiotherapy (RT) Deathl ligand (TRAIL)

Radiotherapy (RT) +

Death ligand (TRAIL)

Ap op tosis stain in g Hy st ol ogy st ai ni ng

(19)